WO2014089290A1 - Modification et régulation du génome basées sur crispr - Google Patents

Modification et régulation du génome basées sur crispr Download PDF

Info

Publication number
WO2014089290A1
WO2014089290A1 PCT/US2013/073307 US2013073307W WO2014089290A1 WO 2014089290 A1 WO2014089290 A1 WO 2014089290A1 US 2013073307 W US2013073307 W US 2013073307W WO 2014089290 A1 WO2014089290 A1 WO 2014089290A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
cell
protein
sequence
fusion protein
Prior art date
Application number
PCT/US2013/073307
Other languages
English (en)
Inventor
Fuqiang Chen
Gregory D. Davis
Qiaohua KANG
Scott W. KNIGHT
Original Assignee
Sigma-Aldrich Co. Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=50883989&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2014089290(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Sigma-Aldrich Co. Llc filed Critical Sigma-Aldrich Co. Llc
Priority to US14/649,777 priority Critical patent/US20160017366A1/en
Priority to CA2891347A priority patent/CA2891347C/fr
Priority to PL18160519T priority patent/PL3363902T3/pl
Priority to PL18156734T priority patent/PL3360964T3/pl
Priority to CN201380072477.4A priority patent/CN105142669B/zh
Priority to KR1020187001934A priority patent/KR102006880B1/ko
Priority to KR1020237015558A priority patent/KR20230070065A/ko
Priority to EP16183724.0A priority patent/EP3138911B1/fr
Priority to EP18160519.7A priority patent/EP3363902B1/fr
Priority to PL16183725T priority patent/PL3138912T3/pl
Priority to IL300199A priority patent/IL300199A/en
Priority to EP16183725.7A priority patent/EP3138912B1/fr
Priority to KR1020227043854A priority patent/KR102531576B1/ko
Priority to SG11201503824SA priority patent/SG11201503824SA/en
Priority to BR112015012375A priority patent/BR112015012375A2/pt
Priority to DK13859964T priority patent/DK2928496T3/da
Priority to EP19201769.7A priority patent/EP3611263A1/fr
Priority to KR1020207023229A priority patent/KR102243092B1/ko
Priority to KR1020217011309A priority patent/KR102479178B1/ko
Priority to AU2013355214A priority patent/AU2013355214B2/en
Priority to KR1020157013843A priority patent/KR101844123B1/ko
Priority to ES13859964T priority patent/ES2757325T3/es
Priority to JP2015545838A priority patent/JP6620018B2/ja
Priority to EP19189913.7A priority patent/EP3617309A3/fr
Priority to PL13859964T priority patent/PL2928496T3/pl
Priority to CN201810574719.4A priority patent/CN108913676B/zh
Priority to KR1020197022305A priority patent/KR102145760B1/ko
Priority to EP13859964.2A priority patent/EP2928496B1/fr
Priority to PL16183724T priority patent/PL3138911T3/pl
Priority to EP18156734.8A priority patent/EP3360964B1/fr
Publication of WO2014089290A1 publication Critical patent/WO2014089290A1/fr
Priority to IL238856A priority patent/IL238856B/en
Priority to HK16106396.9A priority patent/HK1218389A1/zh
Priority to US15/188,902 priority patent/US20160298132A1/en
Priority to US15/188,909 priority patent/US20160298133A1/en
Priority to US15/188,927 priority patent/US20160298136A1/en
Priority to US15/188,931 priority patent/US20160298137A1/en
Priority to US15/188,899 priority patent/US20160298125A1/en
Priority to US15/188,924 priority patent/US10745716B2/en
Priority to US15/188,933 priority patent/US20160298138A1/en
Priority to US15/188,911 priority patent/US10731181B2/en
Priority to US15/342,976 priority patent/US20170073705A1/en
Priority to US15/456,204 priority patent/US20170191082A1/en
Priority to AU2017204031A priority patent/AU2017204031B2/en
Priority to IL257178A priority patent/IL257178B/en
Priority to AU2018229489A priority patent/AU2018229489B2/en
Priority to AU2019201344A priority patent/AU2019201344C1/en
Priority to IL267598A priority patent/IL267598B/en
Priority to US16/654,613 priority patent/US20200140897A1/en
Priority to US16/943,792 priority patent/US20210079427A1/en
Priority to US16/943,767 priority patent/US20210207173A1/en
Priority to AU2020230243A priority patent/AU2020230243B2/en
Priority to AU2020230246A priority patent/AU2020230246B2/en
Priority to AU2020273316A priority patent/AU2020273316B2/en
Priority to US17/398,648 priority patent/US20210388396A1/en
Priority to AU2022200330A priority patent/AU2022200330B2/en
Priority to IL291129A priority patent/IL291129B2/en
Priority to AU2023216829A priority patent/AU2023216829A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21004—Type II site-specific deoxyribonuclease (3.1.21.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates targeted genome modification.
  • the disclosure relates to RNA-guided endonucleases or fusion proteins comprising CRISPR/Cas-like protein and methods of using said proteins to modify or regulate targeted chromosomal sequences.
  • Targeted genome modification is a powerful tool for genetic manipulation of eukaryotic cells, embryos, and animals.
  • exogenous sequences can be integrated at targeted genomic locations and/or specific endogenous chromosomal sequences can be deleted, inactivated, or modified.
  • Current methods rely on the use of engineered nuclease enzymes, such as, for example, zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs).
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • These chimeric nucleases contain programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain.
  • Each new genomic target however, requires the design of a new ZFN or TALEN comprising a novel sequence-specific DNA-binding module.
  • these custom designed nucleases tend to be costly and time-consuming to prepare.
  • the specificities of ZFNs and TALENS are such
  • RNA-guided endonuclease wherein the endonuclease comprises at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific nucleotide sequence for cleavage.
  • the endonuclease can be derived from a Cas9 protein.
  • the endonuclease can be modified to lack at least one functional nuclease domain.
  • the endonuclease can further comprise a cell-penetrating domain, a marker domain, or both.
  • the endonuclease can be part of a protein-RNA complex comprising the guide RNA.
  • the guide RNA can be a single molecule comprising a 5' region that is complementary to a target site.
  • an isolated nucleic acid encoding any of the RNA-guided endonucleases disclosed herein.
  • the nucleic acid can be codon optimized for translation in mammalian cells, such as, for example, human cells.
  • the nucleic acid sequence encoding the RNA-guided endonuclease can be operably linked to a promoter control sequence, and optionally, can be part of a vector. In other
  • a vector comprising sequence encoding the RNA-guided endonuclease, which can be operably linked to a promoter control sequence can also comprise sequence encoding a guide RNA, which can be operably linked to a promoter control sequence.
  • Another aspect of the present invention encompasses a method for modifying a chromosomal sequence in a eukaryotic cell or embryo.
  • the method comprises introducing into a eukaryotic cell or embryo (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease as defined herein, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide comprising a donor sequence.
  • the method further comprises culturing the cell or embryo such that each guide RNA directs a RNA-guided
  • the RNA-guided endonuclease can be derived from a Cas9 protein.
  • the nucleic acid encoding the RNA-guided endonuclease introduced into the cell or embryo can be mRNA. In a further embodiment, wherein the nucleic acid encoding the RNA-guided endonuclease introduced into the cell or embryo can be DNA.
  • the DNA encoding the RNA-guided endonuclease can be part of a vector that further comprises a sequence encoding the guide RNA.
  • the eukaryotic cell can be a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.
  • the embryo is a non-human one cell animal embryo.
  • a further aspect of the disclosure provides a fusion protein comprising a CRISP R/Cas-like protein or fragment thereof and an effector domain.
  • the fusion protein comprises at least one nuclear localization signal.
  • the effector domain of the fusion protein can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the CRISPR/Cas-like protein of the fusion protein can be derived from a Cas9 protein.
  • the Cas9 protein can be modified to lack at least one functional nuclease domain.
  • the Cas9 protein can be modified to lack all nuclease activity.
  • the effector domain can be a cleavage domain, such as, for example, a Fokl endonuclease domain or a modified Fokl endonuclease domain.
  • one fusion protein can form a dimer with another fusion protein.
  • the dimer can be a homodimer or a heterodimer.
  • the fusion protein can form a heterodimer with a zinc finger nuclease, wherein the cleavage domain of both the fusion protein and the zinc finger nucleases is a Fokl endonuclease domain or a modified Fokl endonuclease domain.
  • the fusion protein comprises a CRISPR/Cas-like protein derived from a Cas9 protein modified to lack all nuclease activity, and the effector domain is a Fokl endonuclease domain or a modified Fokl endonuclease domain.
  • the fusion protein comprises a CRISPR/Cas-like protein derived from a Cas9 protein modified to lack all nuclease activity, and the effector domain can be an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • any of the fusion proteins disclosed herein can comprise at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, and a marker domain. Also provided are isolated nucleic acids encoding any of the fusion proteins provided herein.
  • Still another aspect of the disclosure encompasses a method for modifying a chromosomal sequence or regulating expression of a chromosomal sequence in a cell or embryo.
  • the method comprises introducing into the cell or embryo (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, wherein the fusion protein comprises a CRISPR/Cas-like protein or a fragment thereof and an effector domain, and (b) at least one guide RNA or DNA encoding at least one guide RNA, wherein the guide RNA guides the CRISPR/Cas-like protein of the fusion protein to a targeted site in the chromosomal sequence and the effector domain of the fusion protein modifies the chromosomal sequence or regulates expression of the chromosomal sequence.
  • the CRISPR/Cas-like protein of the fusion protein can be derived from a Cas9 protein.
  • the CRISPR/Cas- like protein of the fusion protein can be modified to lack at least one functional nuclease domain.
  • the CRISPR/Cas-like protein of the fusion protein can be modified to lack all nuclease activity.
  • the method can comprise introducing into the cell or embryo one fusion protein or nucleic acid encoding one fusion protein and two guide RNAs or DNA encoding two guide RNAs, and wherein one double-stranded break is introduced in the chromosomal sequence.
  • the method can comprise introducing into the cell or embryo two fusion proteins or nucleic acid encoding two fusion proteins and two guide RNAs or DNA encoding two guide RNAs, and wherein two double-stranded breaks are introduced in the chromosomal sequence.
  • the method can comprise introducing into the cell or embryo one fusion protein or nucleic acid encoding one fusion protein, one guide RNA or nucleic acid encoding one guide RNA, and one zinc finger nuclease or nucleic acid encoding one zinc finger nuclease, wherein the zinc finger nuclease comprises a Fokl cleavage domain or a modified a Fokl cleavage domain, and wherein one double- stranded break is introduced into the chromosomal sequence.
  • the method can further comprise introducing into the cell or embryo at least one donor polynucleotide.
  • the fusion protein comprises an effector domain chosen from an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain
  • the fusion protein can comprise a Cas9 protein modified to lack all nuclease activity
  • the method can comprise introducing into the cell or embryo one fusion protein or nucleic acid encoding one fusion protein, and one guide RNA or nucleic acid encoding one guide RNA, and wherein the structure or expression of the targeted chromosomal sequence is modified.
  • the eukaryotic cell can be a human cell, a non-human mammalian cell, a stem cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.
  • the embryo is a non-human one cell animal embryo.
  • FIG. 1 diagrams genome modification using protein dimers.
  • A depicts a double stranded break created by a dimer composed of two fusion proteins, each of which comprises a Cas-like protein for DNA binding and a Fokl cleavage domain.
  • B depicts a double stranded break created by a dimer composed of a fusion protein comprising a Cas-like protein and a Fokl cleavage domain and a zinc finger nuclease comprising a zinc finger (ZF) DNA-binding domain and a Fokl cleavage domain.
  • ZF zinc finger
  • FIG. 2 illustrates regulation of gene expression using RNA-guided fusion proteins comprising gene regulatory domains.
  • A depicts a fusion protein comprising a Cas-like protein used for DNA binding and an "A/R" domain that activates or represses gene expression.
  • B diagrams a fusion protein comprising a Cas-like protein for DNA binding and a epigenetic modification domain ("Epi-mod') that affects epigenetic states by covalent modification of proximal DNA or proteins.
  • Epi-mod' epigenetic modification domain
  • (A) depicts a double stranded break created by two RNA-guided endonuclease that have been converted into nickases.
  • (B) depicts two double stranded breaks created by two RNA-guided endonuclease having endonuclease activity.
  • FIG. 4 presents fluorescence-activated cell sorting (FACS) of human K562 cells transfected with Cas9 nucleic acid, Cas9 guiding RNA, and AAVS1 - GFP DNA donor.
  • the Y axis represents the auto fluorescence intensity at a red channel, and the X axis represents the green fluorescence intensity.
  • A K562 cells transfected with 10 ⁇ g of Cas9 mRNA transcribed with an Anti-Reverse Cap Analog, 0.3 nmol of pre-annealed crRNA-tracrRNA duplex, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • B K562 cells transfected 10 ⁇ g of Cas9 mRNA transcribed with an Anti-Reverse Cap Analog, 0.3 nmol of chimeric RNA, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • C K562 cells transfected 10 ⁇ g of Cas9 mRNA that was capped by post-transcription capping reaction, 0.3 nmol of chimeric RNA, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • D K562 cells transfected with 10 ⁇ g of Cas9 plasmid DNA, 5 ⁇ g of U6-chimeric RNA plasmid DNA, and 10 ⁇ g
  • FIG. 5 presents a junction PCR analysis documenting the targeted integration of GFP into the AAVS1 locus in human cells.
  • Lane M 1 kb DNA molecular markers
  • Lane A K562 cells transfected with 10 ⁇ g of Cas9 mRNA transcribed with an Anti-Reverse Cap Analog, 0.3 nmol of pre-annealed crRNA-tracrRNA duplex, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • Lane B K562 cells transfected 10 ⁇ g of Cas9 mRNA transcribed with an Anti-Reverse Cap Analog, 0.3 nmol of chimeric RNA, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • Lane C K562 cells transfected 10 ⁇ g of Cas9 mRNA that was capped by post-transcription capping reaction, 0.3 nmol of chimeric RNA, and 10 ⁇ g of AAVS1 -GFP plasmid DNA
  • Lane C
  • RNA-guided endonucleases which comprise at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific nucleotide sequence for cleavage.
  • nucleic acids encoding the RNA- guided endonucleases as well as methods of using the RNA-guided endonucleases to modify chromosomal sequences of eukaryotic cells or embryos.
  • the RNA-guided endonuclease interacts with specific guide RNAs, each of which directs the
  • RNA-guided endonuclease to a specific targeted site, at which site the RNA-guided endonuclease introduces a double-stranded break that can be repaired by a DNA repair process such that the chromosomal sequence is modified. Since the specificity is provided by the guide RNA, the RNA-based endonuclease is universal and can be used with different guide RNAs to target different genomic sequences. The methods disclosed herein can be used to target and modify specific chromosomal sequences and/or introduce exogenous sequences at targeted locations in the genome of cells or embryos.
  • the targeting is specific with limited off target effects.
  • a fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain.
  • Suitable effector domains include, without limit, cleavage domains, epigenetic modification domains, transcriptional activation domains, and transcriptional repressor domains.
  • Each fusion protein is guided to a specific chromosomal sequence by a specific guide RNA, wherein the effector domain mediates targeted genome
  • the fusion proteins can function as dimers thereby increasing the length of the target site and increasing the likelihood of its uniqueness in the genome (thus, reducing off target effects).
  • endogenous CRISPR systems modify genomic locations based on DNA binding word lengths of approximately 13-20 bp (Cong et al., Science, 339:819-823). At this word size, only 5- 7% of the target sites are unique within the genome (Iseli et al, PLos One 2(6):e579).
  • DNA binding word sizes for zinc finger nucleases typically range from 30-36 bp, resulting in target sites that are approximately 85-87% unique within the human genome.
  • the smaller sized DNA binding sites utilized by CRISPR-based systems limits and complicates design of targeted CRISP-based nucleases near desired locations, such as disease SNPs, small exons, start codons, and stop codons, as well as other locations within complex genomes.
  • the present disclosure not only provides means for expanding the CRISPR DNA binding word length (i.e., so as to limit off-target activity), but further provides CRISPR fusion proteins having modified functionality. According, the disclosed CRISPR fusion proteins have increased target specificity and unique functionality(ies). Also provided herein are methods of using the fusion proteins to modify or regulate expression of targeted chromosomal sequences.
  • RNA-guided endonucleases comprising at least one nuclear localization signal, which permits entry of the endonuclease into the nuclei of eukaryotic cells and embryos such as, for example, non-human one cell embryos.
  • RNA-guided endonucleases also comprise at least one nuclease domain and at least one domain that interacts with a guide RNA.
  • An RNA-guided endonuclease is directed to a specific nucleic acid sequence (or target site) by a guide RNA.
  • the guide RNA interacts with the RNA-guided endonuclease as well as the target site such that, once directed to the target site, the RNA-guided
  • the endonuclease is able to introduce a double-stranded break into the target site nucleic acid sequence. Since the guide RNA provides the specificity for the targeted cleavage, the endonuclease of the RNA-guided endonuclease is universal and can be used with different guide RNAs to cleave different target nucleic acid sequences.
  • RNA-guided endonucleases isolated nucleic acids (i.e., RNA or DNA) encoding the RNA-guided endonucleases, vectors comprising nucleic acids encoding the RNA-guided endonucleases, and protein-RNA complexes comprising the RNA- guided endonuclease plus a guide RNA.
  • isolated nucleic acids i.e., RNA or DNA
  • vectors comprising nucleic acids encoding the RNA-guided endonucleases
  • protein-RNA complexes comprising the RNA- guided endonuclease plus a guide RNA.
  • the RNA-guided endonuclease can be derived from a clustered regularly interspersed short p_alindromic repeats (CRISPR)/CRISPR-associated (Cas) system.
  • CRISPR clustered regularly interspersed short p_alindromic repeats
  • Cas CRISPR-associated
  • the CRISPR/Cas system can be a type I, a type II, or a type III system.
  • Non- limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Casl Od, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Cs
  • the RNA-guided endonuclease is derived from a type II CRISPR/Cas system.
  • the RNA-guided endonuclease is derived from a Cas9 protein.
  • the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis rougevillei,
  • Microscilla marina Burkholderiales bacterium, Polaromonas naphthalenivorans,
  • Polaromonas sp. Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii,
  • Caldicieriosiruptor becscii Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum
  • thermopropionicum Acidithiobacillus caldus, Acidithiobacillus ferrooxidans,
  • chthonoplastes Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or
  • CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs. CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • the CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified
  • the CRISPR/Cas-like protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease i.e., DNase, RNase
  • the CRISPR/Cas-like protein can be truncated to remove domains that are not essential for the function of the fusion protein.
  • the CRISPR/Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.
  • the CRISPR/Cas-like protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the
  • CRISPR/Cas-like protein can be derived from modified Cas9 protein.
  • the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • a Cas9 protein comprises at least two nuclease (i.e., DNase) domains.
  • a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek et al., Science, 337: 816-821 ).
  • the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain).
  • the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • the Cas9-derived protein is able to introduce a nick into a double- stranded nucleic acid (such protein is termed a "nickase"), but not cleave the double- stranded DNA.
  • nickase a double- stranded nucleic acid
  • an aspartate to alanine (D10A) conversion in a RuvC-like domain converts the Cas9-derived protein into a nickase.
  • H840A or H839A a histidine to alanine (H840A or H839A) conversion in a HNH domain converts the Cas9-derived protein into a nickase.
  • Each nuclease domain can be modified using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • the RNA-guided endonuclease disclosed herein comprises at least one nuclear localization signal.
  • an NLS comprises a stretch of basic amino acids. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101 -5105).
  • the NLS can be a monopartite sequence, such as PKKKRKV (SEQ ID NO:1 ) or PKKKRRV (SEQ ID NO:2).
  • the NLS can be a bipartite sequence.
  • the NLS can be KRPAATKKAGQAKKKK (SEQ ID NO:3).
  • the NLS can be located at the N-terminus, the C-terminal, or in an internal location of the RNA- guided endonuclease.
  • the RNA-guided endonuclease can further comprise at least one cell-penetrating domain.
  • the cell-penetrating domain can be a cell-penetrating peptide sequence derived from the HIV-1 TAT protein.
  • the TAT cell-penetrating sequence can be
  • the cell- penetrating domain can be TLM (PLSSIFSRIGDPPKKKRKV; SEQ ID NO:5), a cell- penetrating peptide sequence derived from the human hepatitis B virus.
  • the cell-penetrating domain can be MPG
  • the cell-penetrating domain can be Pep-1
  • KETWWETWWTEWSQPKKKRKV SEQ ID NO:8
  • VP22 a cell penetrating peptide from Herpes simplex virus, or a polyarginine peptide sequence.
  • the cell-penetrating domain can be located at the N-terminus, the C-terminus, or in an internal location of the protein.
  • the RNA-guided endonuclease can also comprise at least one marker domain.
  • marker domains include fluorescent proteins, purification tags, and epitope tags.
  • the marker domain can be a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl ), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl ,), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalamal , GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g. ECFP, Cerulean, CyPet,
  • red fluorescent proteins mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed- Tandem, HcRedl , AsRed2, eqFP61 1 , mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.
  • red fluorescent proteins mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed- Tandem, HcRedl , AsRed2, eqFP61 1 , mRasberry, m
  • the marker domain can be a purification tag and/or an epitope tag.
  • Exemplary tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1 , AU5, E, ECS, E2, FLAG, HA, nus, Softag 1 , Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1 , T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • TRX thioredoxin
  • poly(NANP) poly(NANP)
  • TAP tandem affinity purification
  • the RNA-guided endonuclease may be part of a protein-RNA complex comprising a guide RNA.
  • the guide RNA interacts with the RNA-guided endonuclease to direct the endonuclease to a specific target site, wherein the 5' end of the guide RNA base pairs with a specific protospacer sequence.
  • a fusion protein comprising a CRISP R/Cas-like protein or fragment thereof and an effector domain.
  • the CRISP R/Cas-like protein is directed to a target site by a guide RNA, at which site the effector domain can modify or effect the targeted nucleic acid sequence.
  • the effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the fusion protein can further comprise at least one additional domain chosen from a nuclear localization signal, a cell-penetrating domain, or a marker domain.
  • the fusion protein comprises a CRISPR/Cas-like protein or a fragment thereof.
  • CRISPR/Cas-like proteins are detailed above in section (I).
  • the CRISPR/Cas-like protein can be located at the N-terminus, the C-terminus, or in an internal location of the fusion protein
  • the CRISPR/Cas-like protein of the fusion protein can be derived from a Cas9 protein.
  • the Cas9-derived protein can be wild type, modified, or a fragment thereof.
  • the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain).
  • the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a "nickase"), but not cleave the double-stranded DNA.
  • a nickase such protein is termed a "nickase”
  • an aspartate to alanine (D10A) conversion in a RuvC-like domain converts the Cas9-derived protein into a nickase.
  • a histidine to alanine (H840A or H839A) conversion in a HNH domain converts the Cas9- derived protein into a nickase.
  • both of the RuvC-like nuclease domain and the HNH-like nuclease domain can be modified or eliminated such that the Cas9-derived protein is unable to nick or cleave double stranded nucleic acid.
  • all nuclease domains of the Cas9-derived protein can be modified or eliminated such that the Cas9-derived protein lacks all nuclease activity.
  • any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site- directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • the CRISPR/Cas-like protein of the fusion protein is derived from a Cas9 protein in which all the nuclease domains have been inactivated or deleted.
  • the fusion protein also comprises an effector domain.
  • the effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the effector domain can be located at the N-terminus, the C-terminus, or in an internal location of the fusion protein.
  • the effector domain is a cleavage domain.
  • a "cleavage domain” refers to a domain that cleaves DNA.
  • the cleavage domain can be obtained from any endonuclease or exonuclease.
  • Non-limiting examples of endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.
  • the cleavage domain can be derived from a type ll-S endonuclease.
  • Type ll-S endonucleases cleave DNA at sites that are typically several base pairs away the recognition site and, as such, have separable recognition and cleavage domains. These enzymes generally are monomers that transiently associate to form dimers to cleave each strand of DNA at staggered locations.
  • suitable type ll-S endonucleases include Bfil, Bpml, Bsal, Bsgl, BsmBI, Bsml, BspMI, Fokl, Mboll, and Sapl.
  • the cleavage domain of the fusion protein is a Fokl cleavage domain or a derivative thereof.
  • the type ll-S cleavage can be modified to facilitate dimerization of two different cleavage domains (each of which is attached to a CRISP R/Cas-like protein or fragment thereof).
  • the cleavage domain of Fokl can be modified by mutating certain amino acid residues.
  • amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491 , 496, 498, 499, 500, 531 , 534, 537, and 538 of Fokl cleavage domains are targets for modification.
  • modified cleavage domains of Fokl that form obligate heterodimers include a pair in which a first modified cleavage domain includes mutations at amino acid positions 490 and 538 and a second modified cleavage domain that includes mutations at amino acid positions 486 and 499 (Miller et al., 2007, Nat. Biotechnol, 25:778-785; Szczpek et al., 2007, Nat. Biotechnol, 25:786-793).
  • modified Fokl cleavage domains can include three amino acid changes (Doyon et al. 201 1 , Nat. Methods, 8:74- 81 ).
  • one modified Fokl domain (which is termed ELD) can comprise Q486E, I499L, N496D mutations and the other modified Fokl domain (which is termed KKR) can comprise E490K, I538K, H537R mutations.
  • the effector domain of the fusion protein is a Fokl cleavage domain or a modified Fokl cleavage domain.
  • the Cas9-derived can be modified as discussed herein such that its endonuclease activity is eliminated.
  • the Cas9-derived can be modified by mutating the RuvC and HNH domains such that they no longer possess nuclease activity.
  • the effector domain of the fusion protein can be an epigenetic modification domain.
  • epigenetic modification domains alter histone structure and/or chromosomal structure without altering the DNA sequence. Changes histone and/or chromatin structure can lead to changes in gene expression.
  • epigenetic modification include, without limit, acetylation or methylation of lysine residues in histone proteins, and methylation of cytosine residues in DNA.
  • suitable epigenetic modification domains include histone
  • acetyltansferase domains histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.
  • the HAT domain can be derived from EP300 (i.e., E1 A binding protein p300), CREBBP (i.e., CREB-binding protein), CDY1 , CDY2, CDYL1 , CLOCK, ELP3, ESA1 , GCN5 (KAT2A), HAT1 ,KAT2B, KAT5, MYST1 , MYST2, MYST3, MYST4, NCOA1 , NCOA2, NCOA3, NCOAT, P/CAF, Tip60, TAFII250, or TF3C4.
  • the HAT domain is p300
  • the Cas9-derived can be modified as discussed herein such that its endonuclease activity is eliminated.
  • the Cas9-derived can be modified by mutating the RuvC and HNH domains such that they no longer possess nuclease activity.
  • the effector domain of the fusion protein can be a transcriptional activation domain.
  • a transcriptional activation domain interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and/or activate
  • the transcriptional activation domain can be, without limit, a herpes simplex virus VP16 activation domain, VP64 (which is a tetrameric derivative of VP16), a N FKB p65 activation domain, p53 activation domains 1 and 2, a CREB (cAMP response element binding protein) activation domain, an E2A activation domain, and an NFAT (nuclear factor of activated T-cells) activation domain.
  • the transcriptional activation domain can be Gal4, Gcn4, MLL, Rtg3, Gln3, Oaf1 , Pip2, Pdr1 , Pdr3, Pho4, and Leu3.
  • the transcriptional activation domain may be wild type, or it may be a modified version of the original transcriptional activation domain.
  • the effector domain of the fusion protein is a VP16 or VP64 transcriptional activation domain.
  • the Cas9-derived protein can be modified as discussed herein such that its endonuclease activity is eliminated.
  • the Cas9-derived can be modified by mutating the RuvC and HNH domains such that they no longer possess nuclease activity.
  • the effector domain of the fusion protein can be a transcriptional repressor domain.
  • a transcriptional repressor domain interacts with transcriptional control elements and/or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to decrease and/or terminate transcription of a gene.
  • transcriptional repressor domains include inducible cAMP early repressor (ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1 glycine rich repressor domains, Sp1 -like repressors, E(spl) repressors, ⁇ repressor, and MeCP2.
  • the Cas9-derived protein can be modified as discussed herein such that its endonuclease activity is eliminated.
  • the cas9 can be modified by mutating the RuvC and HNH domains such that they no longer possess nuclease activity.
  • the fusion protein further comprises at least one additional domain.
  • suitable additional domains include nuclear localization signals, cell-penetrating or translocation domains, and marker domains.
  • suitable nuclear localization signals, cell-penetrating domains, and marker domains are presented above in section (I).
  • a dimer comprising at least one fusion protein can form.
  • the dimer can be a homodimer or a heterodimer.
  • the heterodimer comprises two different fusion proteins.
  • the heterodimer comprises one fusion protein and an additional protein.
  • the dimer is a homodimer in which the two fusion protein monomers are identical with respect to the primary amino acid sequence.
  • the Cas9-derived proteins are modified such that their endonuclease activity is eliminated, i.e., such that they have no functional nuclease domains.
  • each fusion protein monomer comprises an identical Cas9 like protein and an identical cleavage domain.
  • the cleavage domain can be any cleavage domain, such as any of the exemplary cleavage domains provided herein.
  • the cleavage domain is a Fokl cleavage domain or a modified Fokl cleavage domain.
  • specific guide RNAs would direct the fusion protein monomers to different but closely adjacent sites such that, upon dimer formation, the nuclease domains of the two monomers would create a double stranded break in the target DNA.
  • the dimer is a heterodimer of two different fusion proteins.
  • the CRISP R/Cas-like protein of each fusion protein can be derived from a different CRISPR/Cas protein or from an orthologous CRISPR/Cas protein from a different bacterial species.
  • each fusion protein can comprise a Cas9-like protein, which Cas9-like protein is derived from a different bacterial species.
  • each fusion protein would recognize a different target site (i.e., specified by the protospacer and/or PAM sequence).
  • the guide RNAs could position the heterodimer to different but closely adjacent sites such that their nuclease domains results in an effective double stranded break in the target DNA.
  • the heterodimer can also have modified Cas9 proteins with nicking activity such that the nicking locations are different.
  • each fusion protein of a heterodimer can have different effector domains.
  • the effector domain is a cleavage domain
  • each fusion protein can contain a different modified cleavage domain.
  • each fusion protein can contain a different modified Fokl cleavage domain, as detailed above in section (ll)(b)(i).
  • the Cas-9 proteins can be modified such that their endonuclease activities are eliminated.
  • the two fusion proteins forming a heterodimer can differ in both the CRISPR/Cas-like protein domain and the effector domain.
  • the homodimer or heterodimer can comprise at least one additional domain chosen from nuclear localization signals (NLSs), cell-penetrating, translocation domains and marker domains, as detailed above.
  • NLSs nuclear localization signals
  • cell-penetrating cell-penetrating
  • translocation domains translocation domains
  • marker domains marker domains
  • one or both of the Cas9-derived proteins can be modified such that its endonuclease activity is eliminated or modified.
  • the heterodimer comprises one fusion protein and an additional protein.
  • the additional protein can be a nuclease.
  • the nuclease is a zinc finger nuclease.
  • a zinc finger nuclease comprises a zinc finger DNA binding domain and a cleavage domain.
  • a zinc finger recognizes and binds three (3) nucleotides.
  • a zinc finger DNA binding domain can comprise from about three zinc fingers to about seven zinc fingers.
  • the zinc finger DNA binding domain can be derived from a naturally occurring protein or it can be engineered. See, for example, Beieri et al. (2002) Nat. Biotechnol. 20:135-141 ; Pabo et al. (2001 ) Ann. Rev.
  • the cleavage domain of the zinc finger nuclease can be any cleavage domain detailed above in section (ll)(b)(i).
  • the cleavage domain of the zinc finger nuclease is a Fokl cleavage domain or a modified Fokl cleavage domain.
  • Such a zinc finger nuclease will dimerize with a fusion protein comprising a Fokl cleavage domain or a modified Fokl cleavage domain.
  • the zinc finger nuclease can comprise at least one additional domain chosen from nuclear localization signals, cell-penetrating or translocation domains, which are detailed above.
  • any of the fusion protein detailed above or a dimer comprising at least one fusion protein may be part of a protein-RNA complex comprising at least one guide RNA.
  • a guide RNA interacts with the CRISPR-CasOlike protein of the fusion protein to direct the fusion protein to a specific target site, wherein the 5' end of the guide RNA base pairs with a specific protospacer sequence.
  • nucleic acids encoding any of the RNA-guided endonucleases or fusion proteins described above in sections (I) and (II), respectively.
  • the nucleic acid can be RNA or DNA.
  • the nucleic acid encoding the RNA-guided endonuclease or fusion protein is mRNA.
  • the m RNA can be 5' capped and/or 3' polyadenylated.
  • the nucleic acid encoding the RNA-guided endonuclease or fusion protein is DNA.
  • the DNA can be present in a vector (see below).
  • the nucleic acid encoding the RNA-guided endonuclease or fusion protein can be codon optimized for efficient translation into protein in the eukaryotic cell or animal of interest.
  • codons can be optimized for expression in humans, mice, rats, hamsters, cows, pigs, cats, dogs, fish, amphibians, plants, yeast, insects, and so forth (see Codon Usage Database at www.kazusa.or.jp/codon/).
  • Programs for codon optimization are available as freeware (e.g., OPTIMIZER at
  • endonuclease or fusion protein can be operably linked to at least one promoter control sequence.
  • the DNA coding sequence can be operably linked to a promoter control sequence for expression in the eukaryotic cell or animal of interest.
  • the promoter control sequence can be constitutive, regulated, or tissue-specific.
  • Suitable constitutive promoter control sequences include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor (EDI )-alpha promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, fragments thereof, or combinations of any of the foregoing.
  • suitable regulated promoter control sequences include without limit those regulated by heat shock, metals, steroids, antibiotics, or alcohol.
  • tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF- ⁇ promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.
  • the promoter sequence can be wild type or it can be modified for more efficient or efficacious expression.
  • the encoding DNA can be operably linked to a CMV promoter for constitutive expression in mammalian cells.
  • the sequence encoding the RNA-guided endonuclease or fusion protein can be operably linked to a promoter sequence that is recognized by a phage RNA polymerase for in vitro mRNA synthesis.
  • the in w ro-tran scribed RNA can be purified for use in the methods detailed below in sections (IV) and (V).
  • the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variation of a T7, T3, or SP6 promoter sequence.
  • the DNA encoding the fusion protein is operably linked to a T7 promoter for in vitro mRNA synthesis using T7 RNA polymerase.
  • the sequence encoding the RNA-guided endonuclease or fusion protein can be operably linked to a promoter sequence for in vitro expression of the RNA-guided endonuclease or fusion protein in bacterial or eukaryotic cells.
  • the expressed protein can be purified for use in the methods detailed below in sections (IV) and (V).
  • Suitable bacterial promoters include, without limit, T7 promoters, lac operon promoters, trp promoters, variations thereof, and combinations thereof.
  • An exemplary bacterial promoter is tac which is a hybrid of trp and lac promoters.
  • suitable eukaryotic promoters are listed above.
  • endonuclease or fusion protein also can be linked to a polyadenylation signal (e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal, etc.) and/or at least one transcriptional termination sequence.
  • a polyadenylation signal e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal, etc.
  • BGH bovine growth hormone
  • sequence encoding the RNA- guided endonuclease or fusion protein also can be linked to sequence encoding at least one nuclear localization signal, at least one cell-penetrating domain, and/or at least one marker domain, which are detailed above in section (I).
  • the DNA encoding the RNA-guided endonuclease or fusion protein can be present in a vector.
  • Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, etc.).
  • the DNA encoding the RNA-guided endonuclease or fusion protein is present in a plasmid vector.
  • suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and variants thereof.
  • the vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3 rd edition, 2001 .
  • additional expression control sequences e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.
  • selectable marker sequences e.g., antibiotic resistance genes
  • the expression vector comprising the sequence encoding the RNA-guided endonuclease or fusion protein can further comprise sequence encoding a guide RNA.
  • the sequence encoding the guide RNA generally is operably linked to at least one transcriptional control sequence for expression of the guide RNA in the cell or embryo of interest.
  • DNA encoding the guide RNA can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III).
  • Pol III RNA polymerase III
  • suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1 , and 7SL RNA promoters.
  • Another aspect of the present disclosure encompasses a method for modifying a chromosomal sequence in a eukaryotic cell or embryo.
  • the method comprises introducing into a eukaryotic cell or embryo (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide comprising a donor sequence.
  • the method further comprises culturing the cell or embryo such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break in the targeted site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified.
  • the method can comprise introducing one RNA-guided endonuclease (or encoding nucleic acid) and one guide RNA (or encoding DNA) into a cell or embryo, wherein the RNA-guided endonuclease introduces one double-stranded break in the targeted chromosomal sequence.
  • the double-stranded break in the chromosomal sequence can be repaired by a non-homologous end-joining (NHEJ) repair process.
  • NHEJ non-homologous end-joining
  • the targeted chromosomal sequence can be modified or inactivated.
  • a single nucleotide change can give rise to an altered protein product, or a shift in the reading frame of a coding sequence can inactivate or "knock out" the sequence such that no protein product is made.
  • the optional donor can give rise to an altered protein product, or a shift in the reading frame of a coding sequence can inactivate or "knock out" the sequence such that no protein product is made.
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence at the targeted site during repair of the double-stranded break.
  • the donor sequence in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted site in the chromosomal sequence, the donor sequence can be exchanged with or integrated into the chromosomal sequence at the targeted site during repair mediated by homology-directed repair process.
  • the donor sequence can be ligated directly with the cleaved chromosomal sequence by a non-homologous repair process during repair of the double-stranded break.
  • Exchange or integration of the donor sequence into the chromosomal sequence modifies the targeted chromosomal sequence or introduces an exogenous sequence into the chromosomal sequence of the cell or embryo.
  • the method can comprise introducing two RNA-guided endonucleases (or encoding nucleic acid) and two guide RNAs (or encoding DNA) into a cell or embryo, wherein the RNA-guided endonucleases introduce two double-stranded breaks in the chromosomal sequence. See FIG. 3B.
  • the two breaks can be within several base pairs, within tens of base pairs, or can be separated by many thousands of base pairs.
  • the resultant double-stranded breaks can be repaired by a non-homologous repair process such that the sequence between the two cleavage sites is lost and/or deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break(s).
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence during repair of the double-stranded breaks by either a homology-based repair process (e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence) or a non-homologous repair process (e.g., in embodiments in which the donor sequence is flanked by compatible overhangs).
  • a homology-based repair process e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence
  • a non-homologous repair process e.g., in embodiments in which the donor sequence is flanked by compatible overhangs.
  • the method can comprise introducing one RNA-guided endonuclease modified to cleave one strand of a double-stranded sequence (or encoding nucleic acid) and two guide RNAs (or encoding DNA) into a cell or embryo, wherein each guide RNA directs the RNA-guided endonuclease to a specific target site, at which site the modified endonuclease cleaves one strand (i.e., nicks) of the double-stranded chromosomal sequence, and wherein the two nicks are in opposite stands and in close enough proximity to constitute a double-stranded break. See FIG. 3A.
  • the resultant double-stranded break can be repaired by a non-homologous repair process such that deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break.
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence during repair of the double-stranded break by either a homology-based repair process (e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence) or a non-homologous repair process (e.g., in embodiments in which the donor sequence is flanked by compatible overhangs).
  • a homology-based repair process e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence
  • a non-homologous repair process e.g., in embodiments in which the donor sequence is flanked by compatible overhangs.
  • the method comprises introducing into a cell or embryo at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal.
  • RNA-guided endonucleases and nucleic acids encoding RNA-guided endonucleases are described above in sections (I) and (III), respectively.
  • the RNA-guided endonuclease can be introduced into the cell or embryo as an isolated protein.
  • the RNA-guided endonuclease can further comprise at least one cell-penetrating domain, which facilitates cellular uptake of the protein.
  • the RNA-guided endonuclease can be introduced into the cell or embryo as an mRNA molecule.
  • the RNA-guided endonuclease can be introduced into the cell or embryo as a DNA molecule.
  • DNA sequence encoding the fusion protein is operably linked to a promoter sequence that will function in the cell or embryo of interest.
  • the DNA sequence can be linear, or the DNA sequence can be part of a vector.
  • the fusion protein can be introduced into the cell or embryo as an RNA-protein complex comprising the fusion protein and the guide RNA.
  • DNA encoding the RNA-guided endonuclease can further comprise sequence encoding a guide RNA.
  • each of the sequences encoding the RNA-guided endonuclease and the guide RNA is operably linked to appropriate promoter control sequence that allows expression of the RNA-guided endonuclease and the guide RNA, respectively, in the cell or embryo.
  • the DNA sequence encoding the RNA-guided endonuclease and the guide RNA can further comprise additional expression control, regulatory, and/or processing sequence(s).
  • the DNA sequence encoding the RNA-guided endonuclease and the guide RNA can be linear or can be part of a vector
  • the method also comprises introducing into a cell or embryo at least one guide RNA or DNA encoding at least one guide RNA.
  • a guide RNA interacts with the RNA-guided endonuclease to direct the endonuclease to a specific target site, at which site the 5' end of the guide RNA base pairs with a specific protospacer sequence in the chromosomal sequence.
  • Each guide RNA comprises three regions: a first region at the 5' end that is complementary to the target site in the chromosomal sequence, a second internal region that forms a stem loop structure, and a third 3' region that remains essentially single-stranded.
  • the first region of each guide RNA is different such that each guide RNA guides a fusion protein to a specific target site.
  • the second and third regions of each guide RNA can be the same in all guide RNAs.
  • the first region of the guide RNA is complementary to sequence (i.e., protospacer sequence) at the target site in the chromosomal sequence such that the first region of the guide RNA can base pair with the target site.
  • the first region of the guide RNA can comprise from about 10 nucleotides to more than about 25 nucleotides.
  • the region of base pairing between the first region of the guide RNA and the target site in the chromosomal sequence can be about 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length.
  • the first region of the guide RNA is about 19, 20, or 21 nucleotides in length.
  • the guide RNA also comprises a second region that forms a secondary structure.
  • the secondary structure comprises a stem (or hairpin) and a loop.
  • the length of the loop and the stem can vary.
  • the loop can range from about 3 to about 10 nucleotides in length
  • the stem can range from about 6 to about 20 base pairs in length.
  • the stem can comprise one or more bulges of 1 to about 10 nucleotides.
  • the overall length of the second region can range from about 16 to about 60 nucleotides in length.
  • the loop is about 4 nucleotides in length and the stem comprises about 12 base pairs.
  • the guide RNA also comprises a third region at the 3' end that remains essentially single-stranded.
  • the third region has no complementarity to any chromosomal sequence in the cell of interest and has no complementarity to the rest of the guide RNA.
  • the length of the third region can vary. In general, the third region is more than about 4 nucleotides in length. For example, the length of the third region can range from about 5 to about 60 nucleotides in length.
  • the combined length of the second and third regions (also called the universal or scaffold region) of the guide RNA can range from about 30 to about 120 nucleotides in length. In one aspect, the combined length of the second and third regions of the guide RNA range from about 70 to about 100 nucleotides in length.
  • the guide RNA comprises a single molecule comprising all three regions.
  • the guide RNA can comprise two separate molecules.
  • the first RNA molecule can comprise the first region of the guide RNA and one half of the "stem" of the second region of the guide RNA.
  • the second RNA molecule can comprise the other half of the "stem” of the second region of the guide RNA and the third region of the guide RNA.
  • the first and second RNA molecules each contain a sequence of nucleotides that are complementary to one another.
  • the first and second RNA molecules each comprise a sequence (of about 6 to about 20 nucleotides) that base pairs to the other sequence to form a functional guide RNA.
  • the guide RNA can be introduced into the cell or embryo as a RNA molecule.
  • the RNA molecule can be transcribed in vitro.
  • the RNA molecule can be chemically synthesized.
  • the guide RNA can be introduced into the cell or embryo as a DNA molecule.
  • the DNA encoding the guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in the cell or embryo of interest.
  • the RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III).
  • Suitable Pol III promoters include, but are not limited to, mammalian U6 or H1 promoters.
  • the RNA coding sequence is linked to a mouse or human U6 promoter.
  • the RNA coding sequence is linked to a mouse or human H1 promoter.
  • the DNA molecule encoding the guide RNA can be linear or circular.
  • the DNA sequence encoding the guide RNA can be part of a vector.
  • Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors.
  • the DNA encoding the RNA-guided endonuclease is present in a plasmid vector.
  • suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and variants thereof.
  • the vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like.
  • each can be part of a separate molecule (e.g., one vector containing fusion protein coding sequence and a second vector containing guide RNA coding sequence) or both can be part of the same molecule (e.g., one vector containing coding (and regulatory) sequence for both the fusion protein and the guide RNA).
  • RNA-guided endonuclease in conjunction with a guide RNA is directed to a target site in the chromosomal sequence, wherein the RNA-guided endonuclease introduces a double-stranded break in the chromosomal sequence.
  • the target site has no sequence limitation except that the sequence is immediately followed (downstream) by a consensus sequence.
  • This consensus sequence is also known as a P/otospacer adjacent motif (PAM).
  • PAM P/otospacer adjacent motif
  • Examples of PAM include, but are not limited to, NGG, NGGNG, and NNAGAAW (wherein N is defined as any nucleotide and W is defined as either A or T).
  • the first region (at the 5' end) of the guide RNA is complementary to the protospacer of the target sequence.
  • the first region of the guide RNA is about 19 to 21 nucleotides in length.
  • the sequence of the target site in the chromosomal sequence is 5'-N 19 .2i-/VGG-3'.
  • the PAM is in italics.
  • the target site can be in the coding region of a gene, in an intron of a gene, in a control region of a gene, in a non-coding region between genes, etc.
  • the gene can be a protein coding gene or an RNA coding gene.
  • the gene can be any gene of interest.
  • the method further comprises introducing at least one donor polynucleotide into the embryo.
  • a donor polynucleotide comprises at least one donor sequence.
  • polynucleotide corresponds to an endogenous or native chromosomal sequence.
  • the donor sequence can be essentially identical to a portion of the
  • the donor sequence can comprise a modified version of the wild type sequence at the targeted site such that, upon integration or exchange with the native sequence, the sequence at the targeted chromosomal location comprises at least one nucleotide change.
  • the change can be an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides, or combinations thereof.
  • the cell or embryo/animal can produce a modified gene product from the targeted chromosomal sequence.
  • the donor sequence of the donor polynucleotide corresponds to an exogenous sequence.
  • an "exogenous" sequence refers to a sequence that is not native to the cell or embryo, or a sequence whose native location in the genome of the cell or embryo is in a different location.
  • the exogenous sequence can comprise protein coding sequence, which can be operably linked to an exogenous promoter control sequence such that, upon integration into the genome, the cell or embryo/animal is able to express the protein coded by the integrated sequence.
  • the exogenous sequence can be integrated into the chromosomal sequence such that its expression is regulated by an endogenous promoter control sequence.
  • the exogenous sequence can be a transcriptional control sequence, another expression control sequence, an RNA coding sequence, and so forth. Integration of an exogenous sequence into a chromosomal sequence is termed a "knock in.”
  • the length of the donor sequence can and will vary.
  • the donor sequence can vary in length from several nucleotides to hundreds of nucleotides to hundreds of thousands of nucleotides.
  • Donor polynucleotide comprising upstream and downstream sequences.
  • the donor sequence in the donor polynucleotide is flanked by an upstream sequence and a downstream sequence, which have substantial sequence identity to sequences located upstream and downstream, respectively, of the targeted site in the chromosomal sequence. Because of these sequence similarities, the upstream and downstream sequences of the donor polynucleotide permit
  • the upstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence upstream of the targeted site.
  • the downstream sequence refers to a nucleic acid sequence that shares substantial sequence identity with a chromosomal sequence downstream of the targeted site.
  • substantially sequence identity refers to sequences having at least about 75% sequence identity.
  • the upstream and downstream sequences in the donor polynucleotide can have about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence upstream or downstream to the targeted site.
  • the upstream and downstream sequences in the donor polynucleotide can have about 95% or 100% sequence identity with chromosomal sequences upstream or downstream to the targeted site.
  • the upstream sequence shares substantial sequence identity with a chromosomal sequence located immediately upstream of the targeted site (i.e., adjacent to the targeted site). In other embodiments, the upstream sequence shares substantial sequence identity with a chromosomal sequence that is located within about one hundred (100) nucleotides upstream from the targeted site. Thus, for example, the upstream sequence can share substantial sequence identity with a chromosomal sequence that is located about 1 to about 20, about 21 to about 40, about 41 to about 60, about 61 to about 80, or about 81 to about 100 nucleotides upstream from the targeted site. In one embodiment, the downstream sequence shares substantial sequence identity with a chromosomal sequence located immediately downstream of the targeted site (i.e., adjacent to the targeted site). In other
  • the downstream sequence shares substantial sequence identity with a chromosomal sequence that is located within about one hundred (100) nucleotides downstream from the targeted site.
  • the downstream sequence can share substantial sequence identity with a chromosomal sequence that is located about 1 to about 20, about 21 to about 40, about 41 to about 60, about 61 to about 80, or about 81 to about 100 nucleotides downstream from the targeted site.
  • Each upstream or downstream sequence can range in length from about 20 nucleotides to about 5000 nucleotides.
  • upstream and downstream sequences can comprise about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides.
  • upstream and downstream sequences can comprise about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600,
  • downstream sequences can range in length from about 50 to about 1500 nucleotides.
  • Donor polynucleotides comprising the upstream and downstream sequences with sequence similarity to the targeted chromosomal sequence can be linear or circular.
  • the donor polynucleotide can be part of a vector.
  • the vector can be a plasmid vector.
  • Donor polynucleotide comprising targeted cleavage site(s).
  • the donor polynucleotide can additionally comprise at least one targeted cleavage site that is recognized by the RNA-guided endonuclease.
  • the targeted cleavage site added to the donor polynucleotide can be placed upstream or
  • the donor sequence can be flanked by targeted cleavage sites such that, upon cleavage by the RNA-guided endonuclease, the donor sequence is flanked by overhangs that are compatible with those in the chromosomal sequence generated upon cleavage by the RNA-guided endonuclease. Accordingly, the donor sequence can be ligated with the cleaved chromosomal sequence during repair of the double stranded break by a nonhomologous repair process.
  • donor polynucleotides comprising the targeted cleavage site(s) will be circular (e.g., can be part of a plasmid vector).
  • Donor polynucleotide comprising a short donor sequence with optional overhangs.
  • the donor polynucleotide can be a linear molecule comprising a short donor sequence with optional short overhangs that are compatible with the overhangs generated by the RNA-guided endonuclease.
  • the donor sequence can be ligated directly with the cleaved chromosomal sequence during repair of the double-stranded break.
  • the donor sequence can be less than about 1 ,000, less than about 500, less than about 250, or less than about 100 nucleotides.
  • the donor polynucleotide can be a linear molecule comprising a short donor sequence with blunt ends. In other iterations, the donor polynucleotide can be a linear molecule comprising a short donor sequence with 5' and/or 3' overhangs. The overhangs can comprise 1 , 2, 3, 4, or 5 nucleotides.
  • the donor polynucleotide will be DNA.
  • the DNA may be single-stranded or double-stranded and/or linear or circular.
  • the donor polynucleotide may be a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the donor polynucleotide comprising the donor sequence can be part of a plasmid vector. In any of these situations, the donor polynucleotide comprising the donor sequence can further comprise at least one additional sequence.
  • RNA-targeted endonuclease(s) (or encoding nucleic acid), the guide RNA(s) (or encoding DNA), and the optional donor polynucleotide(s) can be introduced into a cell or embryo by a variety of means.
  • the cell or embryo is transfected. Suitable transfection methods include calcium phosphate- mediated transfection, nucleofection (or electroporation), cationic polymer transfection (e.g., DEAE-dextran or polyethylenimine), viral transduction, virosome transfection, virion transfection, liposome transfection, cationic liposome transfection,
  • the embryo is a fertilized one-cell stage embryo of the species of interest.
  • the molecules can be injected into the pronuclei of one cell embryos.
  • polynucleotide(s) can be introduced into the cell or embryo simultaneously or
  • RNA-targeted endonuclease(s) or encoding nucleic acid
  • guide RNA(s) or encoding DNA
  • DNA encoding an RNA- targeted endonuclease and DNA encoding a guide RNA are delivered together within the plasmid vector.
  • the method further comprises maintaining the cell or embryo under appropriate conditions such that the guide RNA(s) directs the RNA-guided
  • RNA- guided endonuclease(s) introduce at least one double-stranded break in the
  • a double-stranded break can be repaired by a DNA repair process such that the chromosomal sequence is modified by a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof.
  • the double-stranded break can be repaired via a non-homologous end-joining (NHEJ) repair process.
  • NHEJ non-homologous end-joining
  • sequence at the chromosomal sequence can be modified such that the reading frame of a coding region can be shifted and that the chromosomal sequence is inactivated or "knocked out.”
  • An inactivated protein-coding chromosomal sequence does not give rise to the protein coded by the wild type chromosomal sequence.
  • the double- stranded break can be repaired by a homology-directed repair (HDR) process such that the donor sequence is integrated into the chromosomal sequence.
  • HDR homology-directed repair
  • an exogenous sequence can be integrated into the genome of the cell or embryo, or the targeted chromosomal sequence can be modified by exchange of a modified sequence for the wild type chromosomal sequence.
  • the RNA-guided endonuclease can cleave both the targeted chromosomal sequence and the donor polynucleotide.
  • the linearized donor polynucleotide can be integrated into the chromosomal sequence at the site of the double-stranded break by ligation between the donor polynucleotide and the cleaved chromosomal sequence via a NHEJ process.
  • the short donor sequence can be integrated into the chromosomal sequence at the site of the double-stranded break via a NHEJ process.
  • the integration can proceed via the ligation of blunt ends between the short donor sequence and the chromosomal sequence at the site of the double stranded break.
  • the integration can proceed via the ligation of sticky ends (i.e., having 5' or 3' overhangs) between a short donor sequence that is flanked by overhangs that are compatible with those generated by the RNA-targeting endonuclease in the cleaved chromosomal sequence.
  • the cell is maintained under conditions appropriate for cell growth and/or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651 ; and Lombardo et al (2007) Nat. Biotechnology 25:1298-1306. Those of skill in the art appreciate that methods for culturing cells are known in the art and can and will vary depending on the cell type. Routine optimization may be used, in all cases, to determine the best techniques for a particular cell type.
  • An embryo can be cultured in vitro (e.g., in cell culture). Typically, the embryo is cultured at an appropriate temperature and in appropriate media with the necessary 0 2 /C0 2 ratio to allow the expression of the RNA endonuclease and guide RNA, if necessary. Suitable non-limiting examples of media include M2, M16, KSOM, BMOC, and HTF media. A skilled artisan will appreciate that culture conditions can and will vary depending on the species of embryo. Routine optimization may be used, in all cases, to determine the best culture conditions for a particular species of embryo. In some cases, a cell line may be derived from an in vitro-cultured embryo (e.g., an embryonic stem cell line).
  • an embryo may be cultured in vivo by transferring the embryo into the uterus of a female host.
  • the female host is from the same or similar species as the embryo.
  • the female host is pseudo-pregnant.
  • Methods of preparing pseudo-pregnant female hosts are known in the art.
  • methods of transferring an embryo into a female host are known. Culturing an embryo in vivo permits the embryo to develop and can result in a live birth of an animal derived from the embryo. Such an animal would comprise the modified chromosomal sequence in every cell of the body.
  • the cell can be a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, an insect cell, a plant cell, a yeast cell, or a single cell eukaryotic organism.
  • the embryo is non-human mammalian embryo.
  • the embryos can be a one cell non- human mammalian embryo.
  • Exemplary mammalian embryos, including one cell embryos include without limit mouse, rat, hamster, rodent, rabbit, feline, canine, ovine, porcine, bovine, equine, and primate embryos.
  • the cell can be a stem cell.
  • Suitable stem cells include without limit embryonic stem cells, ES-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells and others.
  • the cell is a mammalian cell.
  • Non-limiting examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NS0 cells, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonic mesenchymal C3H-10T1 /2 cells; mouse carcinoma CT26 cells, mouse prostate DuCuP cells; mouse breast EMT6 cells; mouse hepatoma Hepa1 c1 c7 cells; mouse myeloma J5582 cells; mouse epithelial MTD-1 A cells; mouse myocardial MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5F cells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; rat glioblastoma 9L cells; rat B lymph
  • Another aspect of the present disclosure encompasses a method for modifying a chromosomal sequence or regulating expression of a chromosomal sequence in a cell or embryo.
  • the method comprises introducing into the cell or embryo (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, wherein the fusion protein comprises a CRISPR/Cas-like protein or a fragment thereof and an effector domain, and (b) at least one guide RNA or DNA encoding the guide RNA, wherein the guide RNA guides the CRISPR/Cas-like protein of the fusion protein to a targeted site in the chromosomal sequence and the effector domain of the fusion protein modifies the chromosomal sequence or regulates expression of the chromosomal sequence.
  • Fusion proteins comprising a CRISPR/Cas-like protein or a fragment thereof and an effector domain are detailed above in section (II).
  • the fusion proteins disclosed herein further comprise at least one nuclear localization signal.
  • Nucleic acids encoding fusion proteins are described above in section (III).
  • the fusion protein can be introduced into the cell or embryo as an isolated protein (which can further comprise a cell-penetrating domain).
  • the isolated fusion protein can be part of a protein-RNA complex comprising the guide RNA.
  • the fusion protein can be introduced into the cell or embryo as a RNA molecule (which can be capped and/or polyadenylated).
  • the fusion protein can be introduced into the cell or embryo as a DNA molecule.
  • the fusion protein and the guide RNA can be introduced into the cell or embryo as discrete DNA molecules or as part of the same DNA molecule.
  • DNA molecules can be plasmid vectors.
  • the method further comprises introducing into the cell or embryo at least one zinc finger nuclease.
  • Zinc finger nucleases are described above in section (ll)(d).
  • the method further comprises introducing into the cell or embryo at least one donor polynucleotide. Donor polynucleotides are detailed above in section (IV)(d). Means for introducing molecules into cells or embryos, as well as means for culturing cell or embryos are described above in sections (IV)(e) and (IV)(f), respectively. Suitable cells and embryos are described above in section (IV)(g).
  • the method can comprise introducing into the cell or embryo one fusion protein (or nucleic acid encoding one fusion protein) and two guide RNAs (or DNA encoding two guide RNAs).
  • the two guide RNAs direct the fusion protein to two different target sites in the chromosomal sequence, wherein the fusion protein dimerizes (e.g., form a homodimer) such that the two cleavage domains can introduce a double stranded break into the chromosomal sequence. See FIG. 1A.
  • the double-stranded break in the chromosomal sequence can be repaired by a non-homologous end-joining (NHEJ) repair process.
  • NHEJ non-homologous end-joining
  • a single nucleotide change can give rise to an altered protein product, or a shift in the reading frame of a coding sequence can inactivate or "knock out" the sequence such that no protein product is made.
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence at the targeted site during repair of the double-stranded break.
  • the donor sequence can be exchanged with or integrated into the
  • the donor sequence can be ligated directly with the cleaved chromosomal sequence by a non-homologous repair process during repair of the double-stranded break.
  • Exchange or integration of the donor sequence into the chromosomal sequence modifies the targeted chromosomal sequence or introduces an exogenous sequence into the chromosomal sequence of the cell or embryo.
  • the method can comprise introducing into the cell or embryo two different fusion proteins (or nucleic acid encoding two different fusion proteins) and two guide RNAs (or DNA encoding two guide RNAs).
  • the fusion proteins can differ as detailed above in section (II).
  • Each guide RNA directs a fusion protein to a specific target site in the chromosomal sequence, wherein the fusion proteins dimerize (e.g., form a heterodimer) such that the two cleavage domains can introduce a double stranded break into the chromosomal sequence.
  • the resultant double-stranded breaks can be repaired by a non-homologous repair process such that deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break.
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence during repair of the double-stranded break by either a homology-based repair process (e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence) or a nonhomologous repair process (e.g., in embodiments in which the donor sequence is flanked by compatible overhangs).
  • a homology-based repair process e.g., in embodiments in which the donor sequence is flanked by upstream and downstream sequences having substantial sequence identity with upstream and downstream sequences, respectively, of the targeted sites in the chromosomal sequence
  • nonhomologous repair process e.g., in embodiments in which the donor sequence is flanked by compatible overhangs.
  • the method can comprise introducing into the cell or embryo one fusion protein (or nucleic acid encoding one fusion protein), one guide RNA (or DNA encoding one guide RNA), and one zinc finger nuclease (or nucleic acid encoding the zinc finger nuclease), wherein the zinc finger nuclease comprises a Fokl cleavage domain or a modified Fokl cleavage domain.
  • the guide RNA directs the fusion protein to a specific chromosomal sequence, and the zinc finger nuclease is directed to another
  • the fusion protein and the zinc finger nuclease dimerize such that the cleavage domain of the fusion protein and the cleavage domain of the zinc finger nuclease can introduce a double stranded break into the chromosomal sequence. See FIG. 1 B.
  • the resultant double-stranded breaks can be repaired by a non-homologous repair process such that deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break.
  • the optional donor polynucleotide is not present
  • the resultant double-stranded breaks can be repaired by a non-homologous repair process such that deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break.
  • the optional donor polynucleotide such that deletions of
  • the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence during repair of the double-stranded break by either a homology-based repair process (e.g., in
  • the method can comprise introducing into the cell or embryo one fusion protein (or nucleic acid encoding one fusion protein) and one guide RNA (or DNA encoding one guide RNA).
  • the guide RNA directs the fusion protein to a specific chromosomal sequence, wherein the transcriptional activation domain or a transcriptional repressor domain activates or represses expression, respectively, of the targeted chromosomal sequence. See FIG. 2A.
  • the method can comprise introducing into the cell or embryo one fusion protein (or nucleic acid encoding one fusion protein) and one guide RNA (or DNA encoding one guide RNA).
  • the guide RNA directs the fusion protein to a specific chromosomal sequence, wherein the epigenetic modification domain modifies the structure of the targeted the chromosomal sequence. See FIG. 2A.
  • Epigenetic modifications include acetylation, methylation of histone proteins and/or nucleotide methylation.
  • structural modification of the chromosomal sequence leads to changes in expression of the chromosomal sequence.
  • the present disclosure encompasses genetically modified cells, non-human embryos, and non-human animals comprising at least one chromosomal sequence that has been modified using an RNA-guided endonuclease-mediated or fusion protein-mediated process, for example, using the methods described herein.
  • the disclosure provides cells comprising at least one DNA or RNA molecule encoding an RNA-guided endonuclease or fusion protein targeted to a chromosomal sequence of interest or a fusion protein, at least one guide RNA, and optionally one or more donor polynucleotide(s).
  • the disclosure also provides non-human embryos comprising at least one DNA or RNA molecule encoding an RNA-guided endonuclease or fusion protein targeted to a chromosomal sequence of interest, at least one guide RNA, and optionally one or more donor polynucleotide(s).
  • the present disclosure provides genetically modified non-human animals, non-human embryos, or animal cells comprising at least one modified chromosomal sequence.
  • the modified chromosomal sequence may be modified such that it is (1 ) inactivated, (2) has an altered expression or produces an altered protein product, or (3) comprises an integrated sequence.
  • the chromosomal sequence is modified with an RNA guided endonuclease-mediated or fusion protein-mediated process, using the methods described herein.
  • one aspect of the present disclosure provides a genetically modified animal in which at least one chromosomal sequence has been modified.
  • the genetically modified animal comprises at least one inactivated chromosomal sequence.
  • the modified chromosomal sequence may be inactivated such that the sequence is not transcribed and/or a functional protein product is not produced.
  • a genetically modified animal comprising an inactivated chromosomal sequence may be termed a "knock out” or a "conditional knock out.”
  • the inactivated chromosomal sequence can include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a nonsense mutation (i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced).
  • a deletion mutation i.e., deletion of one or more nucleotides
  • an insertion mutation i.e., insertion of one or more nucleotides
  • a nonsense mutation i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced.
  • the inactivated chromosomal sequence comprises no exogenously introduced sequence.
  • genetically modified animals in which two, three, four, five
  • the modified chromosomal sequence can be altered such that it codes for a variant protein product.
  • a genetically modified animal comprising a modified chromosomal sequence can comprise a targeted point mutation(s) or other modification such that an altered protein product is produced.
  • the chromosomal sequence can be modified such that at least one nucleotide is changed and the expressed protein comprises one changed amino acid residue (missense mutation).
  • the chromosomal sequence can be modified to comprise more than one missense mutation such that more than one amino acid is changed.
  • the chromosomal sequence can be modified to have a three nucleotide deletion or insertion such that the expressed protein comprises a single amino acid deletion or insertion.
  • the altered or variant protein can have altered properties or activities compared to the wild type protein, such as altered substrate specificity, altered enzyme activity, altered kinetic rates, etc.
  • the genetically modified animal can comprise at least one chromosomally integrated sequence.
  • a genetically modified animal comprising an integrated sequence may be termed a "knock in” or a
  • the chromosomally integrated sequence can, for example, encode an orthologous protein, an endogenous protein, or combinations of both.
  • a sequence encoding an orthologous protein or an endogenous protein can be integrated into a chromosomal sequence encoding a protein such that the chromosomal sequence is inactivated, but the exogenous sequence is expressed.
  • the sequence encoding the orthologous protein or endogenous protein may be operably linked to a promoter control sequence.
  • a sequence encoding an orthologous protein or an endogenous protein may be integrated into a chromosomal sequence without affecting expression of a chromosomal sequence.
  • a sequence encoding a protein can be integrated into a "safe harbor" locus, such as the Rosa26 locus, HPRT locus, or AAV locus.
  • a "safe harbor" locus such as the Rosa26 locus, HPRT locus, or AAV locus.
  • the present disclosure also encompasses genetically modified animals in which two, three, four, five, six, seven, eight, nine, or ten or more sequences, including sequences encoding protein(s), are integrated into the genome.
  • the chromosomally integrated sequence encoding a protein can encode the wild type form of a protein of interest or can encode a protein comprising at least one modification such that an altered version of the protein is produced.
  • a chromosomally integrated sequence encoding a protein related to a disease or disorder can comprise at least one modification such that the altered version of the protein produced causes or potentiates the associated disorder.
  • the chromosomally integrated sequence encoding a protein related to a disease or disorder can comprise at least one modification such that the altered version of the protein protects against the development of the associated disorder.
  • the genetically modified animal can be a "humanized" animal comprising at least one chromosomally integrated
  • the functional human protein can have no corresponding ortholog in the genetically modified animal.
  • the wild type animal from which the genetically modified animal is derived may comprise an ortholog corresponding to the functional human protein. In this case, the
  • orthologous sequence in the "humanized” animal is inactivated such that no functional protein is made and the "humanized” animal comprises at least one chromosomally integrated sequence encoding the human protein.
  • the genetically modified animal can comprise at least one modified chromosomal sequence encoding a protein such that the expression pattern of the protein is altered.
  • regulatory regions controlling the expression of the protein such as a promoter or a transcription factor binding site, can be altered such that the protein is over-produced, or the tissue-specific or temporal expression of the protein is altered, or a combination thereof.
  • conditional knockout system includes a Cre-lox recombination system.
  • a Cre-lox recombination system comprises a Cre recombinase enzyme, a site-specific DNA recombinase that can catalyze the
  • telomere sequence between specific sites (lox sites) in a nucleic acid molecule.
  • Methods of using this system to produce temporal and tissue specific expression are known in the art.
  • a genetically modified animal is generated with lox sites flanking a chromosomal sequence.
  • the genetically modified animal comprising the lox-flanked chromosomal sequence can then be crossed with another genetically modified animal expressing Cre recombinase.
  • Progeny animals comprising the lox-flanked chromosomal sequence and the Cre recombinase are then produced, and the lox-flanked chromosomal sequence is recombined, leading to deletion or inversion of the chromosomal sequence encoding the protein.
  • Expression of Cre recombinase can be temporally and conditionally regulated to effect temporally and conditionally regulated recombination of the chromosomal sequence.
  • the genetically modified animal disclosed herein can be heterozygous for the modified chromosomal sequence.
  • the genetically modified animal can be homozygous for the modified chromosomal sequence.
  • the genetically modified animals disclosed herein can be crossbred to create animals comprising more than one modified chromosomal sequence or to create animals that are homozygous for one or more modified chromosomal sequences.
  • two animals comprising the same modified chromosomal sequence can be crossbred to create an animal homozygous for the modified chromosomal sequence.
  • animals with different modified chromosomal sequences can be crossbred to create an animal comprising both modified chromosomal sequences.
  • a f i rst animal comprising an inactivated
  • chromosomal sequence gene "x" can be crossed with a second animal comprising a chromosomally integrated sequence encoding a human gene "X" protein to give rise to "humanized” gene "X” offspring comprising both the inactivated gene "x" chromosomal sequence and the chromosomally integrated human gene "X” sequence.
  • a humanized gene "X” animal can be crossed with a humanized gene ⁇ " animal to create humanized gene X/gene Y offspring.
  • an animal comprising a modified
  • chromosomal sequence can be crossbred to combine the modified chromosomal sequence with other genetic backgrounds.
  • other genetic backgrounds may include wild-type genetic backgrounds, genetic backgrounds with deletion mutations, genetic backgrounds with another targeted integration, and genetic backgrounds with non-targeted integrations.
  • animal refers to a non-human animal.
  • the animal may be an embryo, a juvenile, or an adult.
  • Suitable animals include vertebrates such as mammals, birds, reptiles, amphibians, shellfish, and fish. Examples of suitable mammals include without limit rodents, companion animals, livestock, and primates.
  • rodents include mice, rats, hamsters, gerbils, and guinea pigs.
  • Suitable companion animals include but are not limited to cats, dogs, rabbits, hedgehogs, and ferrets.
  • livestock include horses, goats, sheep, swine, cattle, llamas, and alpacas.
  • Suitable primates include but are not limited to capuchin monkeys, chimpanzees, lemurs, macaques,
  • marmosets tamarins, spider monkeys, squirrel monkeys, and vervet monkeys.
  • birds include chickens, turkeys, ducks, and geese.
  • the animal may be an invertebrate such as an insect, a nematode, and the like.
  • insects include Drosophila and mosquitoes.
  • An exemplary animal is a rat.
  • suitable rat strains include Dahl Salt- Sensitive, Fischer 344, Lewis, Long Evans Hooded, Sprague-Dawley, and Wistar.
  • the animal is not a genetically modified mouse. In each of the foregoing iterations of suitable animals for the invention, the animal does not include exogenously introduced, randomly integrated transposon sequences.
  • a further aspect of the present disclosure provides genetically modified cells or cell lines comprising at least one modified chromosomal sequence.
  • the genetically modified cell or cell line can be derived from any of the genetically modified animals disclosed herein.
  • the chromosomal sequence can be modified in a cell as described herein above (in the paragraphs describing chromosomal sequence modifications in animals) using the methods descried herein.
  • the disclosure also encompasses a lysate of said cells or cell lines.
  • the cells are eukaryotic cells.
  • Suitable host cells include fungi or yeast, such as Pichia, Saccharomyces, or Schizosaccharomyces; insect cells, such as SF9 cells from Spodoptera frugiperda or S2 cells from Drosophila melanogaster; and animal cells, such as mouse, rat, hamster, non-human primate, or human cells.
  • Exemplary cells are mammalian.
  • the mammalian cells can be primary cells. In general, any primary cell that is sensitive to double strand breaks may be used.
  • the cells may be of a variety of cell types, e.g., fibroblast, myoblast, T or B cell, macrophage, epithelial cell, and so forth.
  • the cell line can be any established cell line or a primary cell line that is not yet described.
  • the cell line can be adherent or non-adherent, or the cell line can be grown under conditions that encourage adherent, non-adherent or organotypic growth using standard techniques known to individuals skilled in the art.
  • suitable mammalian cells and cell lines are provided herein in section (IV)(g).
  • the cell can be a stem cell.
  • suitable stem cells are provided in section (IV)(g).
  • the present disclosure also provides a genetically modified non- human embryo comprising at least one modified chromosomal sequence.
  • the chromosomal sequence can be modified in an embryo as described herein above (in the paragraphs describing chromosomal sequence modifications in animals) using the methods descried herein.
  • the embryo is a non-human fertilized one-cell stage embryo of the animal species of interest.
  • Exemplary mammalian embryos, including one cell embryos include without limit, mouse, rat, hamster, rodent, rabbit, feline, canine, ovine, porcine, bovine, equine, and primate embryos.
  • endogenous sequence refers to a chromosomal sequence that is native to the cell.
  • exogenous refers to a sequence that is not native to the cell, or a chromosomal sequence whose native location in the genome of the cell is in a different chromosomal location.
  • a "gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.
  • heterologous refers to an entity that is not endogenous or native to the cell of interest.
  • a heterologous protein refers to a protein that is derived from or was originally derived from an exogenous source, such as an exogenously introduced nucleic acid sequence. In some instances, the heterologous protein is not normally produced by the cell of interest.
  • nucleic acid and “polynucleotide” refer to a
  • deoxyribonucleotide or ribonucleotide polymer in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T.
  • nucleotide refers to deoxyribonucleotides
  • nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs.
  • a nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety.
  • a nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a non-naturally occurring nucleotide.
  • Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines).
  • Nucleotide analogs also include dideoxy nucleotides, 2'-0-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity.
  • the percent identity of two sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482- 489 (1981 ). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
  • a Cas9 gene from Streptococcus pyogenes strain MGAS15252 was optimized with Homo sapiens codon preference to enhance its translation in mammalian cells.
  • the Cas9 gene also was modified by adding a nuclear localization signal PKKKRKV (SEQ ID NO:1 ) at the C terminus for targeting the protein into the nuclei of mammalian cells.
  • Table 1 presents the modified Cas9 amino acid sequence, with the nuclear localization sequence underlined.
  • Table 2 presents the codon optimized, modified Cas9 DNA sequence.
  • the modified Cas9 DNA sequence was placed under the control of cytomegalovirus (CMV) promoter for constituent expression in mammalian cells.
  • CMV cytomegalovirus
  • the modified Cas9 DNA sequence was also placed under the control T7 promoter for in vitro mRNA synthesis with T7 RNA polymerase.
  • In vitro RNA transcription was performed by using MessageMAX T7 ARCA-Capped Message Transcription Kit and T7 mScript Standard mRNA Production System (Cellscript).
  • the adeno-associated virus integration site 1 (AAVS1 ) locus was used as a target for Cas9-mediated human genome modification.
  • the human AAVS1 locus is located in intron 1 (4427 bp) of protein phosphatase 1 , regulatory subunit 12C (PPP1 R12C).
  • Table 3 presents the first exon (shaded gray) and the first intron of PPP1 R12C.
  • the underlined sequence within the intron is the targeted modification site (i.e., AAVS1 locus).
  • Cas9 guide RNAs were designed for targeting the human AAVS1 locus.
  • a 42 nucleotide RNA (referred to herein as a "crRNA" sequence) comprising (5' to 3') a target recognition sequence (i.e., sequence complementary to the non-coding strand of the target sequence) and protospacer sequence; a 85 nucleotide RNA
  • tracrRNA sequence (referred to herein as a "tracrRNA” sequence) comprising 5' sequence with
  • a chimeric RNA comprising nucleotides 1 -32 of the crRNA, a GAAA loop, and nucleotides 19-45 of the tracrRNA were prepared.
  • the crRNA was chemically synthesized by Sigma-Aldrich.
  • the tracrRNA and chimeric RNA were synthesized by in vitro transcription with T7 RNA polymerase using T7-Scribe Standard RNA IVT Kit (Cellscript).
  • the chimeric RNA coding sequence was also placed under the control of human U6 promoter for in vivo transcription in human cells. Table 4 presents the sequences of the guide RNAs.
  • Targeted integration of a GFP protein into the N terminus of PPP1 R12C was used to monitor Cas9-mediated genome modification.
  • a donor polynucleotide was prepared.
  • the AAVS1 -GFP DNA donor contained a 5' (1 185 bp) AAVS1 locus homologous arm, an RNA splicing receptor, a turbo GFP coding sequence, a 3' transcription terminator, and a 3' (1217 bp) AAVS1 locus homologous arm.
  • Table 5 presents the sequences of the RNA splicing receptor and the GFP coding sequence followed by the 3' transcription terminator.
  • Plasmid DNA was prepared by using GenElute Endotoxin-Free Plasmid Maxiprep Kit (Sigma).
  • Targeted gene integration will result in a fusion protein between the first 107 amino acids of the PPP1 R12C and the turbo GFP.
  • the expected fusion protein contains the first 107 amino acid residues of PPP1 R12C (highlighted in grey) from RNA splicing between the first exon of PPP1 R12C and the engineered splice receptor (see Table 6).
  • the K562 cell line was obtained from American Type Culture Collection (ATCC) and grown in Iscove's Modified Dulbecco's Medium, supplemented with 10% FBS and 2 mM L-glutamine. All media and supplements were obtained from Sigma-Aldrich. Cultures were split one day before transfection (at approximately 0.5 million cells per ml_ before transfection). Cells were transfected with Nucleofector Solution V (Lonza) on a Nucleofector (Lonza) with the T-016 program. Each nucleofection contained approximately 0.6 million cells.
  • Transfection treatments are detailed in Table 7. Cells were grown at 37 °C and 5% CO 2 immediately after nucleofection.
  • FACS Fluorescence-activated cell sorting
  • Genomic DNA was extracted from transfected cells with GenElute Mammalian Genomic DNA Miniprep Kit (Sigma) 12 days after transfection. Genomic DNA was then PCR amplified with a forward primer located outside the 5' homologous arm of the AAVS1 -GFP plasmid donor and a reverse primer located at the 5' region of the GFP.
  • the forward primer was 5'- CCACTCTGTGCTGACCACTCT-3' (SEQ ID NO:18) and reverse primer was 5'- GCGGCACTCGATCTCCA-3' (SEQ ID NO:19).
  • the expected fragment size from the junction PCR was 1388 bp.
  • the amplification was carried out with JumpStart Taq ReadyMix (Sigma), using the following cycling
  • PCR products were resolved on 1 % agarose gel.
  • the mouse Rosa26 locus can be targeted for genome
  • Table 8 presents a portion of the mouse Rosa26 sequence in which potential target sites are shown in bold. Each target site comprises a protospacer.
  • RNAs were designed to target each of the target sites in the mouse Rosa26 locus.
  • the sequences are shown in Table 9, each is 42 nucleotides in length and the 5' region is complementary to the strand that is not presented in Table 8 (i.e., the strand that is complementary to the strand shown in Table 8).
  • the crRNAs were chemically synthesized and pre-annealed to the tracrRNA (SEQ ID NO:13; see Example 2).
  • Pre-annealed crRNA / tracrRNA and in vitro transcribed mRNA encoding modified Cas9 protein (SEQ ID NO. 9; see Example 1 ) can be microinjected into the pronuclei of fertilized mouse embryos.
  • the Cas9 protein cleaves the target site, and the resultant double-stranded break can be repaired via a non-homologous end-joining (NHEJ) repair process.
  • NHEJ non-homologous end-joining
  • the injected embryos can be either incubated at 37 °C, 5% CO 2 overnight or for up to 4 days, followed by genotyping analysis, or the injected embryos can be implanted into recipient female mice such that live born animals can be genotyped.
  • the in w ' fro-incu bated embryos or tissues from live born animals can be screened for the presence of Cas9-induced mutation at the Rosa locus using standard methods.
  • the embryos or tissues from fetus or live-born animals can be harvested for DNA extraction and analysis. DNA ca n b e isolated using standard procedures.
  • the targeted region of the Rosa26 locus can be PCR amplified using appropriate primers.
  • NHEJ is error-prone, deletions of at least one nucleotide, insertions of at least one nucleotide, substitutions of at least one nucleotide, or combinations thereof can occur during the repair of the break. Mutations can be detected using PCR-based genotyping methods, such as Cel-I mismatch assays and DNA sequencing.
  • the Rosa26 locus can be modified in mouse embryos by co- injecting a donor polynucleotide, as detailed above in section (IV)(d), along with the pre- annealed crRNA / tracrRNA and mRNA encoding modified Cas9 as described above in Example 6.
  • a donor polynucleotide as detailed above in section (IV)(d)
  • mRNA encoding modified Cas9 as described above in Example 6.
  • w ' fro-incu bated embryos or tissues from live born animals (as described in Example 6) can be screened for a modified Rosa26 locus using PCR-based genotyping methods, such as RFLP assays, junction PCR, and DNA sequencing.
  • the rat Rosa26 locus can be targeted for genome modifications.
  • Table 10 presents a portion of the rat sequence in which potential target sites are shown in bold. Each target site comprises a protospacer.
  • RNAs were designed to target each of the target sites in the rat Rosa26 locus. The sequences are shown in Table 1 1 , each is 42 nucleotides in length and the 5' region is complementary to the strand that is not presented in Table 10 (i.e., the strand that is complementary to the strand shown in Table 10).
  • the crRNAs were chemically synthesized and pre-annealed to the tracrRNA (SEQ ID NO:13; see Example 2).
  • Pre-annealed crRNA / tracrRNA and in vitro transcribed mRNA encoding modified Cas9 protein (SEQ ID NO. 9; see Example 1 ) can be microinjected into the pronuclei of fertilized rat embryos.
  • the Cas9 protein cleaves the target site, and the resultant double-stranded break can be repaired via a non-homologous end-joining (NHEJ) repair process.
  • NHEJ non-homologous end-joining
  • the injected embryos can be either incubated at 37 °C, 5% CO 2 overnight or for up to 4 days, followed by genotyping analysis, or the injected embryos can be implanted into recipient female mice such that live born animals can be genotyped.
  • the in w ' fro-incu bated embryos or tissues from live born animals can be screened for the presence of Cas9-induced mutation at the Rosa locus using standard methods.
  • the embryos or tissues from fetus or live-born animals can be harvested for DNA extraction and analysis. DNA ca n b e isolated using standard procedures.
  • the targeted region of the Rosa26 locus can be PCR amplified using appropriate primers.
  • the Rosa26 locus can be modified in rat embryos by co-injecting a donor polynucleotide, as detailed above in section (IV)(d), along with the pre-annealed crRNA / tracrRNA and mRNA encoding modified Cas9 as described above in Example 8.
  • a donor polynucleotide as detailed above in section (IV)(d)
  • mRNA encoding modified Cas9 as described above in Example 8.
  • In vitro- incubated embryos or tissues from live born rats (as described in Example 8) can be screened for a modified Rosa26 locus using PCR-based genotyping methods, such as RFLP assays, junction PCR, and DNA sequencing.

Abstract

La présente invention concerne des endonucléases guidées par l'ARN, qui sont manipulées pour être exprimées dans des cellules eucaryotes ou des embryons, et des procédés d'utilisation de l'endonucléase guidée par l'ARN pour cibler une modification du génome dans des cellules eucaryotes ou des embryons. Elle concerne également des protéines de fusion, chaque protéine de fusion comprenant une protéine de type CRISPR/Cas ou un fragment de celle-ci et un domaine effecteur. Le domaine effecteur peut être un domaine de clivage, un domaine de modification épigénétique, un domaine d'activation de la transcription ou un domaine de répresseur de la transcription. Elle concerne également des procédés d'utilisation des protéines de fusion pour modifier une séquence chromosomique ou réguler l'expression d'une séquence chromosomique.
PCT/US2013/073307 2012-12-06 2013-12-05 Modification et régulation du génome basées sur crispr WO2014089290A1 (fr)

Priority Applications (57)

Application Number Priority Date Filing Date Title
US14/649,777 US20160017366A1 (en) 2012-12-06 2013-12-05 Crispr-based genome modification and regulation
CA2891347A CA2891347C (fr) 2012-12-06 2013-12-05 Modification et regulation du genome basees sur crispr
PL18160519T PL3363902T3 (pl) 2012-12-06 2013-12-05 Modyfikacja i regulacja genomu w oparciu o CRISPR
PL18156734T PL3360964T3 (pl) 2012-12-06 2013-12-05 Modyfikacja i regulacja genomu oparta na crispr
CN201380072477.4A CN105142669B (zh) 2012-12-06 2013-12-05 基于crispr的基因组修饰和调控
KR1020187001934A KR102006880B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
KR1020237015558A KR20230070065A (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
EP16183724.0A EP3138911B1 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
EP18160519.7A EP3363902B1 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
PL16183725T PL3138912T3 (pl) 2012-12-06 2013-12-05 Modyfikacja i regulacja genomu oparta na CRISPR
IL300199A IL300199A (en) 2012-12-06 2013-12-05 CRISPR-based genome modification and regulation
EP16183725.7A EP3138912B1 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
KR1020227043854A KR102531576B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
SG11201503824SA SG11201503824SA (en) 2012-12-06 2013-12-05 Crispr-based genome modification and regulation
BR112015012375A BR112015012375A2 (pt) 2012-12-06 2013-12-05 modificação e regulação de genoma à base de crispr
DK13859964T DK2928496T3 (da) 2012-12-06 2013-12-05 Crispr-baseret genommodifikation og -regulering
EP19201769.7A EP3611263A1 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
KR1020207023229A KR102243092B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
KR1020217011309A KR102479178B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
AU2013355214A AU2013355214B2 (en) 2012-12-06 2013-12-05 Crispr-based genome modification and regulation
KR1020157013843A KR101844123B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
ES13859964T ES2757325T3 (es) 2012-12-06 2013-12-05 Modificación y regulación del genoma en base a CRISPR
JP2015545838A JP6620018B2 (ja) 2012-12-06 2013-12-05 Ｃｒｉｓｐｒに基づくゲノム修飾および制御
EP19189913.7A EP3617309A3 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
PL13859964T PL2928496T3 (pl) 2012-12-06 2013-12-05 Modyfikacja i regulacja genomu w oparciu o CRISPR
CN201810574719.4A CN108913676B (zh) 2012-12-06 2013-12-05 基于crispr的基因组修饰和调控
KR1020197022305A KR102145760B1 (ko) 2012-12-06 2013-12-05 Crispr-기초된 유전체 변형과 조절
EP13859964.2A EP2928496B1 (fr) 2012-12-06 2013-12-05 Modification et régulation du génome basées sur crispr
PL16183724T PL3138911T3 (pl) 2012-12-06 2013-12-05 Modyfikacja i regulacja genomu oparta na CRISPR
EP18156734.8A EP3360964B1 (fr) 2012-12-06 2013-12-05 Modification et régulation de génome à base de crispr
IL238856A IL238856B (en) 2012-12-06 2015-05-17 Crispr-based genome modification and regulation
HK16106396.9A HK1218389A1 (zh) 2012-12-06 2016-06-06 基於 的基因組修飾和調控
US15/188,902 US20160298132A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,909 US20160298133A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,927 US20160298136A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,931 US20160298137A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,899 US20160298125A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,924 US10745716B2 (en) 2012-12-06 2016-06-21 CRISPR-based genome modification and regulation
US15/188,933 US20160298138A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,911 US10731181B2 (en) 2012-12-06 2016-06-21 CRISPR-based genome modification and regulation
US15/342,976 US20170073705A1 (en) 2012-12-06 2016-11-03 Crispr-based genome modification and regulation
US15/456,204 US20170191082A1 (en) 2012-12-06 2017-03-10 Crispr-based genome modification and regulation
AU2017204031A AU2017204031B2 (en) 2012-12-06 2017-06-15 Crispr-based genome modification and regulation
IL257178A IL257178B (en) 2012-12-06 2018-01-28 Crispr-based genome modification and regulation
AU2018229489A AU2018229489B2 (en) 2012-12-06 2018-09-13 Crispr-based genome modification and regulation
AU2019201344A AU2019201344C1 (en) 2012-12-06 2019-02-26 Crispr-based genome modification and regulation
IL267598A IL267598B (en) 2012-12-06 2019-06-24 Crispr-based genome modification and regulation
US16/654,613 US20200140897A1 (en) 2012-12-06 2019-10-16 Crispr-based genome modification and regulation
US16/943,792 US20210079427A1 (en) 2012-12-06 2020-07-30 Crispr-based genome modification and regulation
US16/943,767 US20210207173A1 (en) 2012-12-06 2020-07-30 Crispr-based genome modification and regulation
AU2020230243A AU2020230243B2 (en) 2012-12-06 2020-09-08 Crispr-based genome modification and regulation
AU2020230246A AU2020230246B2 (en) 2012-12-06 2020-09-08 Crispr-based genome modification and regulation
AU2020273316A AU2020273316B2 (en) 2012-12-06 2020-11-19 Crispr-based genome modification and regulation
US17/398,648 US20210388396A1 (en) 2012-12-06 2021-08-10 Crispr-based genome modification and regulation
AU2022200330A AU2022200330B2 (en) 2012-12-06 2022-01-19 Crispr-based genome modification and regulation
IL291129A IL291129B2 (en) 2012-12-06 2022-03-06 Crispr-based genome modification and regulation
AU2023216829A AU2023216829A1 (en) 2012-12-06 2023-08-17 Crispr-based genome modification and regulation

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US201261734256P 2012-12-06 2012-12-06
US61/734,256 2012-12-06
US201361758624P 2013-01-30 2013-01-30
US61/758,624 2013-01-30
US201361761046P 2013-02-05 2013-02-05
US61/761,046 2013-02-05
US201361794422P 2013-03-15 2013-03-15
US61/794,422 2013-03-15

Related Child Applications (10)

Application Number Title Priority Date Filing Date
US14/649,777 A-371-Of-International US20160017366A1 (en) 2012-12-06 2013-12-05 Crispr-based genome modification and regulation
US15/188,933 Continuation US20160298138A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,911 Continuation US10731181B2 (en) 2012-12-06 2016-06-21 CRISPR-based genome modification and regulation
US15/188,931 Continuation US20160298137A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,927 Continuation US20160298136A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,924 Continuation US10745716B2 (en) 2012-12-06 2016-06-21 CRISPR-based genome modification and regulation
US15/188,902 Division US20160298132A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,899 Division US20160298125A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/188,909 Division US20160298133A1 (en) 2012-12-06 2016-06-21 Crispr-based genome modification and regulation
US15/342,976 Continuation US20170073705A1 (en) 2012-12-06 2016-11-03 Crispr-based genome modification and regulation

Publications (1)

Publication Number Publication Date
WO2014089290A1 true WO2014089290A1 (fr) 2014-06-12

Family

ID=50883989

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/073307 WO2014089290A1 (fr) 2012-12-06 2013-12-05 Modification et régulation du génome basées sur crispr

Country Status (17)

Country Link
US (15) US20160017366A1 (fr)
EP (11) EP3138909A1 (fr)
JP (6) JP6620018B2 (fr)
KR (7) KR102145760B1 (fr)
CN (3) CN105142669B (fr)
AU (9) AU2013355214B2 (fr)
BR (1) BR112015012375A2 (fr)
CA (3) CA2977152C (fr)
DK (6) DK3138912T3 (fr)
ES (6) ES2757325T3 (fr)
HK (1) HK1218389A1 (fr)
IL (5) IL300199A (fr)
LT (4) LT3138912T (fr)
PL (6) PL3363902T3 (fr)
PT (6) PT2928496T (fr)
SG (4) SG10201800585VA (fr)
WO (1) WO2014089290A1 (fr)

Cited By (251)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2764103A2 (fr) 2012-12-12 2014-08-13 The Broad Institute, Inc. Systèmes crispr-cas et procédés pour modifier l'expression de produits de gène
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
EP2800811A1 (fr) 2012-05-25 2014-11-12 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US20140356958A1 (en) * 2012-12-17 2014-12-04 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
EP2828386A1 (fr) 2012-03-20 2015-01-28 Vilnius University CLIVAGE D'ADN DIRIGÉ PAR ARN PAR LE COMPLEXE CAS9-ARNcr
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
EP2784162B1 (fr) 2012-12-12 2015-04-08 The Broad Institute, Inc. Ingénierie de systèmes, procédés et compositions de guidage optimisé pour manipulation de séquence
WO2015088643A1 (fr) 2013-12-11 2015-06-18 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
US9068179B1 (en) 2013-12-12 2015-06-30 President And Fellows Of Harvard College Methods for correcting presenilin point mutations
EP2922393A1 (fr) 2013-02-27 2015-09-30 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Édition de gène dans l'ovocyte au moyen de cas9 nucléases
US9163284B2 (en) 2013-08-09 2015-10-20 President And Fellows Of Harvard College Methods for identifying a target site of a Cas9 nuclease
EP2931897A2 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
EP2912175A4 (fr) * 2012-10-23 2015-12-09 Toolgen Inc Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation
WO2015188109A1 (fr) 2014-06-06 2015-12-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification d'un locus ciblé
WO2015195621A1 (fr) * 2014-06-16 2015-12-23 The Johns Hopkins University Compositions et procédés pour l'expression d'arn guide de crispr à l'aide du promoteur h1
WO2015200334A1 (fr) * 2014-06-23 2015-12-30 Regeneron Pharmaceuticals, Inc. Assemblage d'adn à médiation par une nucléase
WO2015200805A2 (fr) 2014-06-26 2015-12-30 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétiques ciblées et procédés d'utilisation
US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
US9260752B1 (en) 2013-03-14 2016-02-16 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
WO2016011080A3 (fr) * 2014-07-14 2016-03-03 The Regents Of The University Of California Modulation transcriptionnelle par crispr/cas
WO2016036754A1 (fr) 2014-09-02 2016-03-10 The Regents Of The University Of California Procédés et compositions pour assurer la modification de l'adn cible arn dépendante
US20160090607A1 (en) * 2013-05-15 2016-03-31 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
EP3004349A1 (fr) * 2013-05-29 2016-04-13 Cellectis S.A. Procédé visant à produire un clivage d'adn précis par l'activité de cas9 nickase
WO2016061374A1 (fr) 2014-10-15 2016-04-21 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
JP2016512264A (ja) * 2013-03-15 2016-04-25 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
JP2016512048A (ja) * 2013-03-15 2016-04-25 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ ＣＲＩＳＰＲ／Ｃａｓシステムを使用した植物ゲノム操作
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
EP3011030A1 (fr) * 2013-06-17 2016-04-27 The Broad Institute, Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
WO2016073433A1 (fr) * 2014-11-06 2016-05-12 E. I. Du Pont De Nemours And Company Administration médiée par un peptide d'endonucléase guidée par un arn dans des cellules
WO2016081923A2 (fr) 2014-11-21 2016-05-26 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée utilisant des arn guides appariés
WO2016080795A1 (fr) * 2014-11-19 2016-05-26 기초과학연구원 Procédé de régulation de l'expression génique à l'aide de la protéine cas9 exprimée par deux vecteurs
WO2016083811A1 (fr) 2014-11-27 2016-06-02 Imperial Innovations Limited Procédés d'édition de génome
WO2016100857A1 (fr) 2014-12-19 2016-06-23 Regeneron Pharmaceuticals, Inc. Cellules souches comme modèle du diabète de type 2
US20160186213A1 (en) * 2013-06-17 2016-06-30 The Broad Institute Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US20160186208A1 (en) * 2013-04-16 2016-06-30 Whitehead Institute For Biomedical Research Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
GB2534074A (en) * 2011-12-30 2016-07-13 Caribou Biosciences Inc Modified cascade ribonucleoproteins and uses thereof
JP2016521554A (ja) * 2013-06-04 2016-07-25 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
JP2016521995A (ja) * 2013-06-17 2016-07-28 ザ・ブロード・インスティテュート・インコーポレイテッド ウイルス成分を使用して障害および疾患をターゲティングするためのＣＲＩＳＰＲ−Ｃａｓ系および組成物の送達、使用および治療上の適用
WO2016120480A1 (fr) 2015-01-29 2016-08-04 Meiogenix Procede pour induire des recombinaisons meiotiques ciblees
JP2016523082A (ja) * 2013-06-17 2016-08-08 ザ・ブロード・インスティテュート・インコーポレイテッド 分裂終了細胞の疾患および障害をターゲティングおよびモデリングするための系、方法および組成物の送達、エンジニアリングおよび最適化
JP2016524472A (ja) * 2013-06-17 2016-08-18 ザ・ブロード・インスティテュート・インコーポレイテッド 肝臓ターゲティングおよび治療のためのＣＲＩＳＰＲ−Ｃａｓ系、ベクターおよび組成物の送達および使用
JP2016528894A (ja) * 2013-07-26 2016-09-23 プレジデント アンド フェローズ オブ ハーバード カレッジ ゲノムエンジニアリング
WO2016176617A2 (fr) 2015-04-29 2016-11-03 New York University Procédé pour le traitement de gliomes de haut grade
WO2016178207A1 (fr) 2015-05-04 2016-11-10 Ramot At Tel-Aviv University Ltd. Procédés et kits pour fragmenter l'adn
US9512446B1 (en) 2015-08-28 2016-12-06 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
WO2016205554A1 (fr) * 2015-06-17 2016-12-22 Poseida Therapeutics, Inc. Compositions et procédés permettant de diriger des protéines vers des loci spécifiques dans le génome
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
WO2016210271A1 (fr) * 2015-06-24 2016-12-29 Sigma-Aldrich Co. Llc Modification et régulation génomique dépendantes du cycle cellulaire
US20170007679A1 (en) * 2014-03-25 2017-01-12 Editas Medicine Inc. Crispr/cas-related methods and compositions for treating hiv infection and aids
WO2017027910A1 (fr) 2015-08-14 2017-02-23 The University Of Sydney Inhibition de la connexine 45 pour la thérapie
WO2017040348A1 (fr) 2015-08-28 2017-03-09 The General Hospital Corporation Nucléases crispr-cas9 modifiées
WO2017075335A1 (fr) 2015-10-28 2017-05-04 Voyager Therapeutics, Inc. Expression régulable au moyen d'un virus adéno-associé (vaa)
US20170121693A1 (en) * 2015-10-23 2017-05-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
JP2017518082A (ja) * 2014-06-17 2017-07-06 ポセイダ セラピューティクス， インコーポレイテッド ゲノム中の特異的遺伝子座にタンパク質を指向させるための方法およびその使用
US20170198268A1 (en) * 2014-07-09 2017-07-13 Gen9, Inc. Compositions and Methods for Site-Directed DNA Nicking and Cleaving
JP2017526387A (ja) * 2014-08-06 2017-09-14 トゥールジェン インコーポレイテッド カンピロバクター・ジェジュニｃｒｉｓｐｒ／ｃａｓシステムに由来するｒｇｅｎを使用したゲノム編集
EP3138910B1 (fr) 2012-12-06 2017-09-20 Sigma-Aldrich Co. LLC Modification et régulation de génome à base de crispr
EP3219799A1 (fr) 2016-03-17 2017-09-20 IMBA-Institut für Molekulare Biotechnologie GmbH Expression sgrna crispr conditionnelle
WO2017186718A1 (fr) * 2016-04-25 2017-11-02 Universität Basel Édition d'allèles et ses applications
WO2017201476A1 (fr) 2016-05-20 2017-11-23 Regeneron Pharmaceuticals, Inc. Procédés pour briser la tolérance immunologique à l'aide de multiples arn de guidage
CN107429246A (zh) * 2014-10-31 2017-12-01 麻省理工学院 用于crispr的大规模并行组合遗传学
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9834786B2 (en) 2012-04-25 2017-12-05 Regeneron Pharmaceuticals, Inc. Nuclease-mediated targeting with large targeting vectors
EP3152319A4 (fr) * 2014-06-05 2017-12-27 Sangamo BioSciences, Inc. Méthodes et compositions pour conception de nucléases
EP3272867A1 (fr) * 2016-06-02 2018-01-24 Sigma-Aldrich Co. LLC Utilisation de protéines de liaison à l'adn programmables pour améliorer la modification ciblée du génome
WO2018022967A1 (fr) 2016-07-28 2018-02-01 Regeneron Pharmaceuticals, Inc. Variants de gpr156 et leurs utilisations
WO2018023014A1 (fr) 2016-07-29 2018-02-01 Regeneron Pharmaceuticals, Inc. Souris comprenant des mutations entraînant l'expression de la fibrilline 1 tronquée en c
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US9926546B2 (en) 2015-08-28 2018-03-27 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
WO2018054911A1 (fr) 2016-09-23 2018-03-29 Bayer Cropscience Nv Optimisation ciblée du génome dans des plantes
US9938521B2 (en) 2014-03-10 2018-04-10 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating leber's congenital amaurosis 10 (LCA10)
EP3204399A4 (fr) * 2014-10-09 2018-04-25 Seattle Children's Hospital, dba Seattle Children's Research Institute Longs plasmides poly (a) et méthodes d'introduction de longues séquences poly(a) dans le plasmide
WO2018073237A1 (fr) 2016-10-17 2018-04-26 The University Court Of The University Of Edinburgh Porcs comprenant le gène cd163 modifié et procédés associés
US10011850B2 (en) 2013-06-21 2018-07-03 The General Hospital Corporation Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
WO2018136758A1 (fr) 2017-01-23 2018-07-26 Regeneron Pharmaceuticals, Inc. Variants de hsd17b13 et utilisations correspondantes
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
WO2018195545A2 (fr) 2017-04-21 2018-10-25 The General Hospital Corporation Variantes de cpf1 (cas12a) à spécificité pam modifiée
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
EP3008186B1 (fr) 2013-06-14 2018-11-28 Cellectis Procédés d'édition de génome non transgénique dans des plantes
WO2018218206A1 (fr) 2017-05-25 2018-11-29 The General Hospital Corporation Architectures d'éditeur de base bipartite (bbe) et édition de doigt de zinc de type ii-c-cas9
WO2018226560A1 (fr) 2017-06-05 2018-12-13 Regeneron Pharmaceuticals, Inc. Variants de b4galt1 et utilisations associées
US10166255B2 (en) 2015-07-31 2019-01-01 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
WO2019006034A1 (fr) 2017-06-27 2019-01-03 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus asgr1 humanisé
WO2019028029A1 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Évaluation de la recombinaison induite par crispr/cas avec un acide nucléique donneur exogène in vivo
WO2019028032A1 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Cellules souches embryonnaires de souris transgéniques cas et souris et leurs utilisations
WO2019028023A2 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour évaluer une disruption ou une excision médiée par crispr/cas et une recombinaison induite par crispr/cas à l'aide d'un acide nucléique donneur exogène in vivo
US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
WO2019050948A1 (fr) * 2017-09-05 2019-03-14 Regeneron Pharmaceuticals, Inc. Administration d'un système d'édition génique comportant une seule particule rétrovirale et méthodes de génération et d'utilisation
WO2019051033A1 (fr) 2017-09-07 2019-03-14 Regeneron Pharmaceuticals, Inc. Variants du membre 1 de la famille 14 des transporteurs de solutés (slc14a1) et leurs utilisations
WO2019050899A1 (fr) 2017-09-06 2019-03-14 Regeneron Pharmaceuticals, Inc. Variants apparentés au récepteur de l'interleukine 1 d'immunoglobuline unique (sigirr) et leurs utilisations
US10240145B2 (en) * 2015-11-25 2019-03-26 The Board Of Trustees Of The Leland Stanford Junior University CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer
EP3460063A1 (fr) 2013-12-11 2019-03-27 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
WO2019067875A1 (fr) 2017-09-29 2019-04-04 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé et procédés d'utilisation
WO2019079238A1 (fr) 2017-10-16 2019-04-25 Regeneron Pharmaceuticals, Inc. Variants de cornulin (crnn) et leurs utilisations
WO2019094735A1 (fr) 2017-11-10 2019-05-16 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant une mutation de slc30a8 et procédés d'utilisation
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
WO2019108983A1 (fr) 2017-11-30 2019-06-06 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus trkb humanisé
EP3494997A1 (fr) * 2012-07-25 2019-06-12 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles, outils de perturbation du génome et leurs applications
WO2019126578A1 (fr) * 2017-12-20 2019-06-27 Poseida Therapeutics, Inc. Compositions et procédés permettant de diriger des protéines vers des loci spécifiques dans le génome
WO2019123014A1 (fr) * 2017-12-22 2019-06-27 G+Flas Life Sciences Molécules d'ingénierie génomique chimère et procédés
US10337001B2 (en) 2014-12-03 2019-07-02 Agilent Technologies, Inc. Guide RNA with chemical modifications
EP3362571A4 (fr) * 2015-10-13 2019-07-10 Duke University Ingénierie génomique avec systèmes crispr de type i dans des cellules eucaryotes
WO2019138083A1 (fr) 2018-01-12 2019-07-18 Basf Se Gène sous-jacent au nombre de qtl d'épillets par épi de blé sur le chromosome 7a
US10377998B2 (en) 2013-12-12 2019-08-13 The Broad Institute, Inc. CRISPR-CAS systems and methods for altering expression of gene products, structural information and inducible modular CAS enzymes
US10385359B2 (en) 2013-04-16 2019-08-20 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
US10415024B2 (en) 2012-11-16 2019-09-17 Poseida Therapeutics, Inc. Site-specific enzymes and methods of use
WO2019183123A1 (fr) 2018-03-19 2019-09-26 Regeneron Pharmaceuticals, Inc. Modulation de la transcription chez des animaux à l'aide de systèmes crispr/cas
US10428319B2 (en) 2017-06-09 2019-10-01 Editas Medicine, Inc. Engineered Cas9 nucleases
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
WO2019217803A1 (fr) 2018-05-10 2019-11-14 Auxolytic Ltd. Procédés et compositions de thérapie génique utilisant des cellules régulables auxotrophes
US10494621B2 (en) 2015-06-18 2019-12-03 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
WO2019234750A1 (fr) 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Procédés de régénération et de transformation de cannabis
WO2019234754A1 (fr) 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Constructions d'acide nucléique et procédés d'utilisation de celles-ci
US10519457B2 (en) 2013-08-22 2019-12-31 E I Du Pont De Nemours And Company Soybean U6 polymerase III promoter and methods of use
US10526589B2 (en) 2013-03-15 2020-01-07 The General Hospital Corporation Multiplex guide RNAs
US10550372B2 (en) 2013-12-12 2020-02-04 The Broad Institute, Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
WO2020030783A2 (fr) 2018-08-10 2020-02-13 Vib Vzw Moyens et procédés de tolérance à la sécheresse dans des cultures
WO2020041313A1 (fr) * 2018-08-21 2020-02-27 Sigma-Aldrich Co. Llc Régulation à la baisse de la voie de détection de l'adn cytosolique
WO2020056122A1 (fr) 2018-09-13 2020-03-19 Regeneron Pharmaceuticals, Inc. Rat à invalidation génique de facteur h de complément en tant que modèle de glomérulopathie c3
US10640789B2 (en) 2013-06-04 2020-05-05 President And Fellows Of Harvard College RNA-guided transcriptional regulation
EP3653048A1 (fr) 2014-12-19 2020-05-20 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée par ciblage multiple en une seule étape
WO2020123377A1 (fr) 2018-12-10 2020-06-18 Neoimmunetech, Inc. Cellules défaillantes nrf-2 et utilisations de celles-ci
WO2020131632A1 (fr) 2018-12-20 2020-06-25 Regeneron Pharmaceuticals, Inc. Expansion de répétition à médiation par nucléase
WO2020128478A1 (fr) 2018-12-19 2020-06-25 King's College London Procédés et compositions immunothérapeutiques
US10696986B2 (en) 2014-12-12 2020-06-30 The Board Institute, Inc. Protected guide RNAS (PGRNAS)
WO2020163396A1 (fr) 2019-02-04 2020-08-13 The General Hospital Corporation Variants d'éditeur de base d'adn adénine avec édition d'arn hors cible réduite
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
WO2020169974A1 (fr) 2019-02-19 2020-08-27 King's College London Récepteurs antigéniques chimériques sensibles à l'hypoxie
US10767193B2 (en) 2018-02-15 2020-09-08 Sigma-Aldrich Co. Llc Engineered CAS9 systems for eukaryotic genome modification
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
US10781444B2 (en) 2013-06-17 2020-09-22 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
WO2020190927A1 (fr) 2019-03-18 2020-09-24 Regeneron Pharmaceuticals, Inc. Plate-forme de criblage crispr/cas pour révéler des vulnérabilités génétiques associées à une agrégation de tau
WO2020190932A1 (fr) 2019-03-18 2020-09-24 Regeneron Pharmaceuticals, Inc. Plate-forme de criblage crispr/cas pour identifier des modificateurs génétiques de l'ensemencement ou de l'agrégation de la protéine tau
WO2020206139A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus facteur 12 de coagulation humanisé
WO2020206134A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés pour l'introduction sans cicatrice de modifications ciblées dans des vecteurs de ciblage
WO2020206162A1 (fr) 2019-04-03 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour l'insertion de séquences de codage d'anticorps dans un locus d'hébergement sûr
WO2020212541A1 (fr) 2019-04-16 2020-10-22 University Of Nottingham Souches fongiques, leur production et leurs utilisations
WO2020229241A1 (fr) 2019-05-10 2020-11-19 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
US10851357B2 (en) 2013-12-12 2020-12-01 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
WO2020247812A1 (fr) 2019-06-07 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'albumine humanisé
WO2020247452A1 (fr) 2019-06-04 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé ayant une mutation bêta-slip et procédés d'utilisation
WO2020252340A1 (fr) 2019-06-14 2020-12-17 Regeneron Pharmaceuticals, Inc. Modèles de tauopathie
CN112105732A (zh) * 2018-05-10 2020-12-18 先正达参股股份有限公司 用于多核苷酸的靶向编辑的方法和组合物
US10912797B2 (en) 2016-10-18 2021-02-09 Intima Bioscience, Inc. Tumor infiltrating lymphocytes and methods of therapy
US10930367B2 (en) 2012-12-12 2021-02-23 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for Cas enzymes or CRISPR-Cas systems for target sequences and conveying results thereof
US10934536B2 (en) 2018-12-14 2021-03-02 Pioneer Hi-Bred International, Inc. CRISPR-CAS systems for genome editing
US10947517B2 (en) 2019-02-15 2021-03-16 Sigma-Aldrich Co. Llc CRISPR/Cas fusion proteins and systems
WO2021050398A1 (fr) * 2019-09-10 2021-03-18 The Regents Of The University Of California Plate-forme de criblage de létalité synthétique pour cellules subissant un alt
WO2021050940A1 (fr) 2019-09-13 2021-03-18 Regeneron Pharmaceuticals, Inc. Modulation de la transcription chez des animaux à l'aide de systèmes crispr/cas administrés par des nanoparticules lipidiques
WO2021048316A1 (fr) 2019-09-12 2021-03-18 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
WO2021069387A1 (fr) 2019-10-07 2021-04-15 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
GB202103131D0 (en) 2021-03-05 2021-04-21 Biosystems Tech Limited Method for preparation of research organisms
WO2021092513A1 (fr) 2019-11-08 2021-05-14 Regeneron Pharmaceuticals, Inc. Stratégies crispr-vaa pour la thérapie du rétinoschisis juvénile lié à l'x
WO2021108363A1 (fr) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Régulation à la hausse médiée par crispr/cas d'un allèle ttr humanisé
US11028388B2 (en) 2014-03-05 2021-06-08 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa
WO2021110582A1 (fr) 2019-12-03 2021-06-10 Basf Se Molécules d'acides nucléiques régulatrices pour améliorer l'expression génique dans des plantes
US11033584B2 (en) 2017-10-27 2021-06-15 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
US11078483B1 (en) 2016-09-02 2021-08-03 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
US11078481B1 (en) 2016-08-03 2021-08-03 KSQ Therapeutics, Inc. Methods for screening for cancer targets
WO2021154791A1 (fr) 2020-01-28 2021-08-05 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus pnpla3 humanisé et procédés d'utilisation
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
WO2021158883A1 (fr) 2020-02-07 2021-08-12 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus klkb1 humanisé et procédés d'utilisation
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
US11111521B2 (en) 2011-12-22 2021-09-07 President And Fellows Of Harvard College Compositions and methods for analyte detection
WO2021178556A1 (fr) 2020-03-04 2021-09-10 Regeneron Pharmaceuticals, Inc. Méthodes et compositions pour la sensibilisation de cellules tumorales à une thérapie immunitaire
WO2021195079A1 (fr) 2020-03-23 2021-09-30 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé affichant une mutation v30m et méthodes d'utilisation
WO2021202938A1 (fr) 2020-04-03 2021-10-07 Creyon Bio, Inc. Apprentissage automatique fondé sur des oligonucléotides
US11141493B2 (en) 2014-03-10 2021-10-12 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
US11149267B2 (en) 2013-10-28 2021-10-19 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
US11155795B2 (en) 2013-12-12 2021-10-26 The Broad Institute, Inc. CRISPR-Cas systems, crystal structure and uses thereof
US11180793B2 (en) 2015-04-24 2021-11-23 Editas Medicine, Inc. Evaluation of Cas9 molecule/guide RNA molecule complexes
WO2021263146A2 (fr) 2020-06-26 2021-12-30 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'ace2 humanisé
GB202118058D0 (en) 2021-12-14 2022-01-26 Univ Warwick Methods to increase yields in crops
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11242525B2 (en) 2014-03-26 2022-02-08 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating sickle cell disease
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11299767B2 (en) 2013-03-12 2022-04-12 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11306309B2 (en) 2015-04-06 2022-04-19 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation
EP3988649A1 (fr) * 2013-09-18 2022-04-27 Kymab Limited Procédés, cellules et organismes
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
EP3998344A1 (fr) * 2014-10-09 2022-05-18 Life Technologies Corporation Oligonucléotides crispr et édition de gènes
US11339437B2 (en) 2014-03-10 2022-05-24 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
WO2022120022A1 (fr) 2020-12-02 2022-06-09 Regeneron Pharmaceuticals, Inc. Lignées cellulaires de biocapteurs crispr sam et procédés d'utilisation associés
US11384360B2 (en) 2012-06-19 2022-07-12 Regents Of The University Of Minnesota Gene targeting in plants using DNA viruses
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11407985B2 (en) 2013-12-12 2022-08-09 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for genome editing
US11427817B2 (en) 2015-08-25 2022-08-30 Duke University Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases
US11434491B2 (en) 2018-04-19 2022-09-06 The Regents Of The University Of California Compositions and methods for gene editing
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11466271B2 (en) 2017-02-06 2022-10-11 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
WO2022240846A1 (fr) 2021-05-10 2022-11-17 Sqz Biotechnologies Company Méthodes pour administrer des molécules d'édition génomique au noyau ou au cytosol d'une cellule et leurs utilisations
US11512311B2 (en) 2016-03-25 2022-11-29 Editas Medicine, Inc. Systems and methods for treating alpha 1-antitrypsin (A1AT) deficiency
WO2022251644A1 (fr) 2021-05-28 2022-12-01 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2022256437A1 (fr) 2021-06-02 2022-12-08 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542554B2 (en) 2015-11-03 2023-01-03 President And Fellows Of Harvard College Method and apparatus for volumetric imaging
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11560568B2 (en) 2014-09-12 2023-01-24 E. I. Du Pont De Nemours And Company Generation of site-specific-integration sites for complex trait loci in corn and soybean, and methods of use
US11566263B2 (en) 2016-08-02 2023-01-31 Editas Medicine, Inc. Compositions and methods for treating CEP290 associated disease
US11578312B2 (en) 2015-06-18 2023-02-14 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
WO2023064924A1 (fr) 2021-10-14 2023-04-20 Codiak Biosciences, Inc. Cellules productrices modifiées pour la production de vésicules extracellulaires
WO2023077053A2 (fr) 2021-10-28 2023-05-04 Regeneron Pharmaceuticals, Inc. Procédés et compositions associés à crispr/cas pour la désactivation de c5
WO2023081847A1 (fr) 2021-11-04 2023-05-11 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus cacng1 modifié
US20230151345A1 (en) * 2014-10-24 2023-05-18 Life Technologies Corporation Compositions and methods for enhancing homologous recombination
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
WO2023108047A1 (fr) 2021-12-08 2023-06-15 Regeneron Pharmaceuticals, Inc. Modèle de maladie impliquant une myociline mutante et ses utilisations
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
EP4198124A1 (fr) 2021-12-15 2023-06-21 Versitech Limited Cas9-nucléases modifiées et leur procédé d'utilisation
WO2023122506A1 (fr) 2021-12-20 2023-06-29 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant des loci ace2 et tmprss humanisés
WO2023129974A1 (fr) 2021-12-29 2023-07-06 Bristol-Myers Squibb Company Génération de lignées de cellules avec site d'intégration
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US11713485B2 (en) 2016-04-25 2023-08-01 President And Fellows Of Harvard College Hybridization chain reaction methods for in situ molecular detection
WO2023150620A1 (fr) 2022-02-02 2023-08-10 Regeneron Pharmaceuticals, Inc. Insertion de transgène médiée par crispr dans des cellules néonatales
WO2023150798A1 (fr) 2022-02-07 2023-08-10 Regeneron Pharmaceuticals, Inc. Compositions et procédés permettant de définir des délais de traitement optimaux dans le cadre d'une maladie lysosomale
WO2023150181A1 (fr) 2022-02-01 2023-08-10 President And Fellows Of Harvard College Méthodes et compositions pour le traitement du cancer
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
WO2023212677A2 (fr) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification de zones de sécurité extragéniques spécifiques de tissu pour des approches de thérapie génique
US11814624B2 (en) 2017-06-15 2023-11-14 The Regents Of The University Of California Targeted non-viral DNA insertions
WO2023220603A1 (fr) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vecteurs et procédés de production d'anticorps in vivo
WO2023225665A1 (fr) 2022-05-19 2023-11-23 Lyell Immunopharma, Inc. Polynucléotides ciblant nr4a3 et leurs utilisations
WO2023235725A2 (fr) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Agents thérapeutiques à base de crispr pour une maladie d'expansion de répétition c9orf72
WO2023235726A2 (fr) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Agents thérapeutiques d'interférence crispr pour une maladie d'expansion de répétition c9orf72
US11851690B2 (en) 2017-03-14 2023-12-26 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
US11859219B1 (en) 2016-12-30 2024-01-02 Flagship Pioneering Innovations V, Inc. Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
WO2024003579A1 (fr) 2022-06-30 2024-01-04 University Of Newcastle Upon Tyne Prévention de la récurrence d'une maladie dans une thérapie de remplacement mitochondrial
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11884915B2 (en) 2021-09-10 2024-01-30 Agilent Technologies, Inc. Guide RNAs with chemical modification for prime editing
WO2024026488A2 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus de récepteur de transférrine modifié
WO2024026474A1 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024031053A1 (fr) 2022-08-05 2024-02-08 Regeneron Pharmaceuticals, Inc. Variants de tdp-43 résistants à l'agrégation
US11898176B2 (en) 2012-04-27 2024-02-13 Duke University Genetic correction of mutated genes
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11913015B2 (en) 2017-04-17 2024-02-27 University Of Maryland, College Park Embryonic cell cultures and methods of using the same
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024073606A1 (fr) 2022-09-28 2024-04-04 Regeneron Pharmaceuticals, Inc. Récepteurs modifiés résistants aux anticorps pour améliorer des thérapies à base de cellules

Families Citing this family (104)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114230675A (zh) * 2013-06-05 2022-03-25 杜克大学 Rna指导的基因编辑和基因调节
WO2015066119A1 (fr) 2013-10-30 2015-05-07 North Carolina State University Compositions et méthodes liées à un système crispr-cas de type ii chez lactobacillus buchneri
US10787654B2 (en) * 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
CN111705365A (zh) 2014-02-11 2020-09-25 科罗拉多州立大学董事会（法人团体） Crispr支持的多路基因组工程化
WO2015126927A2 (fr) 2014-02-18 2015-08-27 Duke University Compositions destinées à l'inactivation de la réplication d'un virus et leurs procédés de fabrication et d'utilisation
CA2944978C (fr) 2014-04-08 2024-02-13 North Carolina State University Procedes et compositions pour la repression dirigee par l'arn de la transcription au moyen de genes associes a crispr
WO2016033298A1 (fr) 2014-08-28 2016-03-03 North Carolina State University Nouvelles protéines cas9 et éléments de guidage pour le ciblage de l'adn et l'édition du génome
CN107532142A (zh) * 2014-11-11 2018-01-02 应用干细胞有限公司 使用同源重组改造间充质干细胞
JP2018516563A (ja) 2015-05-29 2018-06-28 ノース カロライナ ステート ユニバーシティＮｏｒｔｈ Ｃａｒｏｌｉｎａ Ｓｔａｔｅ Ｕｎｉｖｅｒｓｉｔｙ Ｃｒｉｓｐｒ核酸を用いて、細菌、古細菌、藻類、および、酵母をスクリーニングする方法
EP3303585A4 (fr) 2015-06-03 2018-10-31 Board of Regents of the University of Nebraska Traitement des données d'une séquence d'adn utilisant un adn monocaténaire
CN115537421A (zh) 2015-06-15 2022-12-30 北卡罗来纳州立大学 有效递送核酸和基于rna的抗微生物剂的方法和组合物
KR101795999B1 (ko) 2015-09-25 2017-11-09 전남대학교산학협력단 Crispr/cas9 시스템을 이용한 베타2-마이크로글로불린 유전자 제거용 시발체
KR101745863B1 (ko) 2015-09-25 2017-06-12 전남대학교산학협력단 Crispr/cas9 시스템을 이용한 프로히비틴2 유전자 제거용 시발체
WO2017058751A1 (fr) 2015-09-28 2017-04-06 North Carolina State University Méthodes et compositions pour agents antimicrobiens spécifiques d'une séquence
PT3846375T (pt) 2015-10-22 2022-12-28 Ericsson Telefon Ab L M Métodos e aparelho relativos ao melhoramento seletivo de sinais de rádio
CN106893739A (zh) 2015-11-17 2017-06-27 香港中文大学 用于靶向基因操作的新方法和系统
WO2017112620A1 (fr) 2015-12-22 2017-06-29 North Carolina State University Méthodes et compositions pour l'administration d'agents antimicrobiens à base de crispr
MX2018008344A (es) 2016-01-11 2018-12-06 Univ Leland Stanford Junior Proteinas quimericas y metodos para regular la expresion genica.
BR112018013663A2 (pt) 2016-01-11 2019-01-22 Univ Leland Stanford Junior proteínas quiméricas e métodos de imunoterapia
SG11201808920RA (en) * 2016-04-14 2018-11-29 Boco Silicon Valley Inc Genome editing of human neural stem cells using nucleases
RU2771383C2 (ru) * 2016-06-03 2022-05-04 Энсэрм (Энститю Насьональ Де Ла Санте Э Де Ла Решерш Медикаль) Контролируемая пищевым рационом экспрессия нуклеиновой кислоты, кодирующей нуклеазу cas9, и ее применения
WO2017213896A1 (fr) * 2016-06-03 2017-12-14 Temple University - Of The Commonwealth System Of Higher Education Régulation à contre-réaction du vih-1 par une stratégie d'édition génique
US10337051B2 (en) 2016-06-16 2019-07-02 The Regents Of The University Of California Methods and compositions for detecting a target RNA
US11293021B1 (en) 2016-06-23 2022-04-05 Inscripta, Inc. Automated cell processing methods, modules, instruments, and systems
AU2017280353B2 (en) 2016-06-24 2021-11-11 Inscripta, Inc. Methods for generating barcoded combinatorial libraries
WO2018013720A1 (fr) * 2016-07-12 2018-01-18 Washington University Incorporation d'étiquettes de séquence poly-lysine codées par polya interne et leurs variations pour la régulation accordable de la synthèse de protéines dans des cellules bactériennes et eucaryotes
US20190185850A1 (en) * 2016-08-20 2019-06-20 Avellino Lab Usa, Inc. Single guide rna/crispr/cas9 systems, and methods of use thereof
WO2018048827A1 (fr) * 2016-09-07 2018-03-15 Massachusetts Institute Of Technology Assemblage d'adn à base d'endonucléases guidées par arn
CN106636197B (zh) * 2016-09-22 2019-09-03 南京市妇幼保健院 一种定向敲降斑马鱼基因组中多拷贝基因的方法
BR112019006384A2 (pt) * 2016-09-30 2019-06-25 Univ California enzimas modificadoras de ácido nucleico guiadas por rna e métodos de uso das mesmas
EP3525831A4 (fr) * 2016-10-11 2020-06-03 Stemgenics, Inc. Nanoparticules fonctionnalisées avec des outils d'édition de gène et procédés associés
EP3535392B1 (fr) * 2016-11-02 2024-03-27 Universität Basel Variants de surface cellulaire discernables sur le plan immunologique destinés à être utilisés en thérapie cellulaire
CN110300802A (zh) * 2016-12-23 2019-10-01 基础科学研究院 用于动物胚胎碱基编辑的组合物和碱基编辑方法
CN106978438B (zh) * 2017-02-27 2020-08-28 北京大北农生物技术有限公司 提高同源重组效率的方法
US11834670B2 (en) 2017-04-19 2023-12-05 Global Life Sciences Solutions Usa Llc Site-specific DNA modification using a donor DNA repair template having tandem repeat sequences
US9982279B1 (en) 2017-06-23 2018-05-29 Inscripta, Inc. Nucleic acid-guided nucleases
US10011849B1 (en) 2017-06-23 2018-07-03 Inscripta, Inc. Nucleic acid-guided nucleases
ES2913457T3 (es) 2017-06-30 2022-06-02 Inscripta Inc Métodos, módulos, instrumentos y sistemas de procesamiento celular automatizados
CA3066798A1 (fr) * 2017-07-31 2019-02-07 Sigma Aldrich Co. Llc Arn guide synthetique pour systemes d'activateurs crispr/cas
CN111373040A (zh) * 2017-08-09 2020-07-03 本森希尔股份有限公司 修饰基因组的组合物和方法
US10738327B2 (en) 2017-08-28 2020-08-11 Inscripta, Inc. Electroporation cuvettes for automation
JP6985508B2 (ja) 2017-09-30 2021-12-22 インスクリプタ， インコーポレイテッド フロースルーエレクトロポレーション機器編成
US11634716B2 (en) 2017-10-16 2023-04-25 University of Pittsburgh—of the Commonwealth System of Higher Education Genetically modified mesenchymal stem cells for use in cardiovascular prosthetics
JP7423520B2 (ja) * 2017-11-16 2024-01-29 アストラゼネカ・アクチエボラーグ Ｃａｓ９ベースノックイン方針の効力を改善するための組成物及び方法
EP3743516A4 (fr) * 2018-01-26 2021-10-13 The Children's Medical Center Corporation Ciblage d'& xc9;l& xc9;ments r& xc9;gulateurs distaux de bcl11a avec une fusion cas9-cas9 pour la r& xc9;induction de l'h& xc9;moglobine f& x152;tale
AU2019241967A1 (en) 2018-03-29 2020-11-19 Inscripta, Inc. Automated control of cell growth rates for induction and transformation
WO2019200004A1 (fr) 2018-04-13 2019-10-17 Inscripta, Inc. Instruments de traitement cellulaire automatisés comprenant des cartouches de réactif
US10858761B2 (en) 2018-04-24 2020-12-08 Inscripta, Inc. Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells
US10501738B2 (en) 2018-04-24 2019-12-10 Inscripta, Inc. Automated instrumentation for production of peptide libraries
US10557216B2 (en) 2018-04-24 2020-02-11 Inscripta, Inc. Automated instrumentation for production of T-cell receptor peptide libraries
WO2019209912A2 (fr) * 2018-04-27 2019-10-31 Seattle Children's Hospital (dba Seattle Children's Research Institute) Édition thérapeutique du génome dans le syndrome hyper-igm lié au sexe
WO2019222545A1 (fr) 2018-05-16 2019-11-21 Synthego Corporation Méthodes et systèmes de conception et d'utilisation d'arn guide
CN108624622A (zh) * 2018-05-16 2018-10-09 湖南艾佳生物科技股份有限公司 一种基于CRISPR-Cas9系统构建的能分泌小鼠白细胞介素-6的基因工程细胞株
EP3575402A1 (fr) * 2018-06-01 2019-12-04 Algentech SAS Ciblage de gènes
EP4159872A1 (fr) * 2018-06-25 2023-04-05 Bionano Genomics, Inc. Marquage de l'adn
EP4070802A1 (fr) 2018-06-30 2022-10-12 Inscripta, Inc. Instruments, modules et procédés de détection améliorée de séquences éditées dans des cellules vivantes
CN112805379A (zh) * 2018-08-03 2021-05-14 比姆医疗股份有限公司 多效应核碱基编辑器和使用其修饰核酸靶序列的方法
US10532324B1 (en) 2018-08-14 2020-01-14 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
US11142740B2 (en) 2018-08-14 2021-10-12 Inscripta, Inc. Detection of nuclease edited sequences in automated modules and instruments
US10752874B2 (en) 2018-08-14 2020-08-25 Inscripta, Inc. Instruments, modules, and methods for improved detection of edited sequences in live cells
KR102158428B1 (ko) * 2018-08-16 2020-09-22 (주)라트바이오 인위적 뉴클레아제를 생산하는 형질전환 배아 및 형질전환 동물의 제작 방법
CN109055379B (zh) * 2018-09-10 2022-04-15 石铭 一种转基因鸡输卵管生物反应器的制备方法
KR102121817B1 (ko) * 2018-09-12 2020-06-26 한국화학연구원 Crispr 편집 기술을 이용한 재조합 항원을 발현시키는 벡터 및 이를 동시에 다중 삽입시키는 방법
US10711267B2 (en) 2018-10-01 2020-07-14 North Carolina State University Recombinant type I CRISPR-Cas system
GB2611929B (en) 2018-10-16 2023-11-22 Blueallele Corp Methods for targeted insertion of DNA in genes
CN113227368B (zh) 2018-10-22 2023-07-07 因思科瑞普特公司 工程化酶
US11214781B2 (en) 2018-10-22 2022-01-04 Inscripta, Inc. Engineered enzyme
US20220380740A1 (en) * 2018-10-24 2022-12-01 The Broad Institute, Inc. Constructs for improved hdr-dependent genomic editing
US20220048962A1 (en) * 2019-01-04 2022-02-17 The University Of Chicago Systems and methods for modulating rna
US20220136004A1 (en) * 2019-01-11 2022-05-05 Chan Zuckerberg Biohub, Inc. Targeted In Vivo Genome Modification
WO2020168234A1 (fr) * 2019-02-14 2020-08-20 Metagenomi Ip Technologies, Llc Enzymes ayant des domaines ruvc
MX2021010559A (es) 2019-03-07 2021-12-15 Univ California Polipéptidos efectores de crispr-cas y métodos de uso de estos.
CN113631713A (zh) 2019-03-25 2021-11-09 因思科瑞普特公司 酵母中的同时多重基因组编辑
US11001831B2 (en) 2019-03-25 2021-05-11 Inscripta, Inc. Simultaneous multiplex genome editing in yeast
CN113939593A (zh) 2019-06-06 2022-01-14 因思科瑞普特公司 用于递归的核酸指导的细胞编辑的处治
US10907125B2 (en) 2019-06-20 2021-02-02 Inscripta, Inc. Flow through electroporation modules and instrumentation
CA3139124C (fr) 2019-06-21 2023-01-31 Inscripta, Inc. Mutations de conception rationnelle a l'echelle du genome conduisant a une production amelioree de lysine dans e. coli
US10927385B2 (en) 2019-06-25 2021-02-23 Inscripta, Inc. Increased nucleic-acid guided cell editing in yeast
CN110628825A (zh) * 2019-10-14 2019-12-31 上海捷易生物科技有限公司 一种依赖nhej的报告基因敲入组合物及其使用方法
WO2021102059A1 (fr) 2019-11-19 2021-05-27 Inscripta, Inc. Procédés pour augmenter l'édition observée dans des bactéries
WO2021118626A1 (fr) 2019-12-10 2021-06-17 Inscripta, Inc. Nouvelles nucléases mad
US10704033B1 (en) 2019-12-13 2020-07-07 Inscripta, Inc. Nucleic acid-guided nucleases
AU2020404580A1 (en) 2019-12-16 2022-06-23 BASF Agricultural Solutions Seed US LLC Improved genome editing using paired nickases
EP4069851A4 (fr) 2019-12-18 2023-11-22 Inscripta, Inc. Dosages de complémentation en cascade/dcas3 pour la détection in vivo de cellules modifiées par une nucléase guidée par un acide nucléique
EP4087600A4 (fr) * 2020-01-09 2024-01-24 Pioneer Hi Bred Int Échange de gène en deux étapes
US10689669B1 (en) 2020-01-11 2020-06-23 Inscripta, Inc. Automated multi-module cell processing methods, instruments, and systems
CN115135370A (zh) 2020-01-27 2022-09-30 因思科瑞普特公司 电穿孔模块和仪器
EP4127156A4 (fr) 2020-03-31 2024-03-27 Metagenomi Inc Systèmes crispr de classe ii, type ii
US20210332388A1 (en) 2020-04-24 2021-10-28 Inscripta, Inc. Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells
US11787841B2 (en) 2020-05-19 2023-10-17 Inscripta, Inc. Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli
CN111849986A (zh) * 2020-07-24 2020-10-30 江苏集萃药康生物科技有限公司 一种减少CRISPR-Cas9基因编辑中双链DNA片段串联的方法及其应用
WO2022060749A1 (fr) 2020-09-15 2022-03-24 Inscripta, Inc. Édition par crispr pour incorporer des plaques d'amarrage d'acide nucléique dans des génomes de cellules vivantes
US11512297B2 (en) 2020-11-09 2022-11-29 Inscripta, Inc. Affinity tag for recombination protein recruitment
WO2022133139A2 (fr) * 2020-12-16 2022-06-23 Administrators Of The Tulane Educational Fund Adipocytes wnt+, exosomes d'adipocyte wnt+, et leurs procédés de fabrication et d'utilisation
AU2021415461A1 (en) 2021-01-04 2023-08-17 Inscripta, Inc. Mad nucleases
EP4274890A1 (fr) 2021-01-07 2023-11-15 Inscripta, Inc. Nucléases mad
US11884924B2 (en) 2021-02-16 2024-01-30 Inscripta, Inc. Dual strand nucleic acid-guided nickase editing
WO2023077148A1 (fr) 2021-11-01 2023-05-04 Tome Biosciences, Inc. Plateforme de construction unique pour administration simultanée d'une machinerie d'édition de gène et d'une cargaison d'acide nucléique
WO2023122764A1 (fr) 2021-12-22 2023-06-29 Tome Biosciences, Inc. Co-administration d'une construction d'éditeur génique et d'un patron donneur
WO2023205744A1 (fr) 2022-04-20 2023-10-26 Tome Biosciences, Inc. Compositions d'insertion de gènes programmable
WO2023215831A1 (fr) 2022-05-04 2023-11-09 Tome Biosciences, Inc. Compositions d'arn guide pour insertion de gène programmable
WO2023225670A2 (fr) 2022-05-20 2023-11-23 Tome Biosciences, Inc. Insertion de gène programmable ex vivo
WO2023250384A2 (fr) * 2022-06-22 2023-12-28 The Regents Of The University Of California Polypeptides effecteurs crispr-cas et leurs procédés d'utilisation
WO2024020587A2 (fr) 2022-07-22 2024-01-25 Tome Biosciences, Inc. Insertion de gènes programmable par des cellules souches pléiopluripotentes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055728A1 (en) * 2006-08-04 2010-03-04 Georgia State University Research Foundation, Inc. Enzyme sensors, methods for preparing and using such sensors, and methods of detecting protease activity
US20110217739A1 (en) * 2008-11-06 2011-09-08 University Of Georgia Research Foundation, Inc. Cas6 polypeptides and methods of use
WO2012012738A1 (fr) * 2010-07-23 2012-01-26 Sigma-Aldrich Co., Llc Modifications du génome à l'aide d'endonucléases de ciblage et d'acides nucléiques simples brins
US20120192298A1 (en) * 2009-07-24 2012-07-26 Sigma Aldrich Co. Llc Method for genome editing
WO2013142578A1 (fr) * 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr

Family Cites Families (163)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4952496A (en) 1984-03-30 1990-08-28 Associated Universities, Inc. Cloning and expression of the gene for bacteriophage T7 RNA polymerase
WO1988008450A1 (fr) 1987-05-01 1988-11-03 Birdwell Finlayson Therapie genetique pour troubles du metabolisme
US5350689A (en) 1987-05-20 1994-09-27 Ciba-Geigy Corporation Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells
US5767367A (en) 1990-06-23 1998-06-16 Hoechst Aktiengesellschaft Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation
US7150982B2 (en) 1991-09-09 2006-12-19 Third Wave Technologies, Inc. RNA detection assays
FR2763797B1 (fr) * 1997-05-30 1999-07-16 Tabacs & Allumettes Ind Cigarette a tres faible taux de goudron presentant un gout de tabac comparable a celui d'une cigarette classique a plus fort taux de goudron
US20040186071A1 (en) 1998-04-13 2004-09-23 Bennett C. Frank Antisense modulation of CD40 expression
US20020182673A1 (en) 1998-05-15 2002-12-05 Genentech, Inc. IL-17 homologous polypedies and therapeutic uses thereof
CA2361191A1 (fr) 1999-02-03 2000-08-10 The Children's Medical Center Corporation Reparation genique a induction de clivage d'adn bicatenaire en un site cible chromosomique
US8183339B1 (en) * 1999-10-12 2012-05-22 Xigen S.A. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
WO2002026967A2 (fr) 2000-09-25 2002-04-04 Thomas Jefferson University Correction de gene cible au moyen d'oligodesoxynucleotides monocatenaires
CA2425303A1 (fr) 2000-10-27 2002-05-02 John Telford Acides nucleiques et proteines derives des groupes de streptocoques a et b
US7033744B2 (en) * 2001-03-16 2006-04-25 Naoya Kobayashi Method for proliferating a liver cell, a liver cell obtained thereby, and use thereof
IL159756A0 (en) 2001-07-12 2004-06-20 Univ Massachusetts IN VIVO PRODUCTION OF SMALL INTERFERING RNAs THAT MEDIATE GENE SILENCING
US20060253913A1 (en) 2001-12-21 2006-11-09 Yue-Jin Huang Production of hSA-linked butyrylcholinesterases in transgenic mammals
CA2474486C (fr) 2002-01-23 2013-05-14 The University Of Utah Research Foundation Mutagenese chromosomique ciblee au moyen de nucleases en doigt a zinc
WO2003080809A2 (fr) 2002-03-21 2003-10-02 Sangamo Biosciences, Inc. Procedes et compositions permettant d'utiliser des endonucleases a doigts de zinc pour ameliorer la recombinaison homologue
AU2003224897A1 (en) 2002-04-09 2003-10-27 Kenneth L. Beattie Oligonucleotide probes for genosensor chips
CA2488668C (fr) * 2002-06-06 2013-02-26 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Modification de recombinaison et de reparation d'adn
AU2003298574B2 (en) 2002-09-05 2008-04-24 California Institute Of Technology Use of chimeric nucleases to stimulate gene targeting
DE10260805A1 (de) * 2002-12-23 2004-07-22 Geneart Gmbh Verfahren und Vorrichtung zum Optimieren einer Nucleotidsequenz zur Expression eines Proteins
AU2004263865B2 (en) 2003-08-08 2007-05-17 Sangamo Therapeutics, Inc. Methods and compositions for targeted cleavage and recombination
WO2005070948A1 (fr) 2004-01-23 2005-08-04 Intronn, Inc. Correction de defauts genetiques lies a l'alpha-1-antitrypsine par transepissage d'arn a mediation par spliceosome
US7972854B2 (en) 2004-02-05 2011-07-05 Sangamo Biosciences, Inc. Methods and compositions for targeted cleavage and recombination
US20050220796A1 (en) 2004-03-31 2005-10-06 Dynan William S Compositions and methods for modulating DNA repair
US7919277B2 (en) 2004-04-28 2011-04-05 Danisco A/S Detection and typing of bacterial strains
EP2316942B1 (fr) * 2004-12-22 2021-04-21 Alnylam Pharmaceuticals, Inc. Sequences vhb et vhc conservees utilisees pour un silençage genique
US7892224B2 (en) 2005-06-01 2011-02-22 Brainlab Ag Inverse catheter planning
US7534819B2 (en) 2005-06-10 2009-05-19 University Of Washington Compositions and methods for intracellular delivery of biotinylated cargo
US20060282289A1 (en) 2005-06-14 2006-12-14 Healthmatch Solutions, Llc System and method for health care financing
US20100055793A1 (en) * 2005-07-25 2010-03-04 Johns Hopkins University Site-specific modification of the human genome using custom-designed zinc finger nucleases
AU2006272634B2 (en) * 2005-07-26 2013-01-24 Sangamo Therapeutics, Inc. Targeted integration and expression of exogenous nucleic acid sequences
US10022457B2 (en) 2005-08-05 2018-07-17 Gholam A. Peyman Methods to regulate polarization and enhance function of cells
PL2325332T3 (pl) 2005-08-26 2013-03-29 Dupont Nutrition Biosci Aps Zastosowanie genów związanych z CRISPR (genów CAS)
KR100877824B1 (ko) * 2005-11-11 2009-01-12 한국생명공학연구원 Ｅ2ｅｐｆ ｕｃｐ-ｖｈｌ 상호작용 및 그 용도
EP2522748A1 (fr) * 2006-03-02 2012-11-14 The Ohio State University Profil d'expression de micro-ARN associé au cancer du pancréas
WO2007106690A2 (fr) 2006-03-15 2007-09-20 Siemens Healthcare Diagnostics Inc. Analogues de nucléobase dégénérée
SG170784A1 (en) * 2006-03-28 2011-05-30 Novartis Ag Covalently-linked complexes of hiv tat and env proteins
EP2016183B1 (fr) 2006-05-10 2011-06-15 Deinove Procédé d'ingénierie chromosomique utilisant un nouveau système de réparation d'adn
US9816140B2 (en) 2006-05-19 2017-11-14 Dupont Nutrition Biosciences Aps Tagged microorganisms and methods of tagging
JP5551432B2 (ja) 2006-05-25 2014-07-16 サンガモ バイオサイエンシーズ， インコーポレイテッド 遺伝子不活性化のための方法と組成物
AU2007258872A1 (en) 2006-06-16 2007-12-21 Danisco A/S Bacterium
SI2126130T1 (sl) 2007-03-02 2015-10-30 Dupont Nutrition Biosciences Aps Kulture z izboljšano fagno odpornostjo
GB0806086D0 (en) 2008-04-04 2008-05-14 Ulive Entpr Ltd Dendrimer polymer hybrids
US8546553B2 (en) 2008-07-25 2013-10-01 University Of Georgia Research Foundation, Inc. Prokaryotic RNAi-like system and methods of use
KR20160015400A (ko) 2008-08-22 2016-02-12 상가모 바이오사이언스 인코포레이티드 표적화된 단일가닥 분할 및 표적화된 통합을 위한 방법 및 조성물
CA2737180C (fr) 2008-09-15 2019-02-19 Children's Medical Center Corporation Modulation de bcl11a pour le traitement d'hemoglobinopathies
US20100076057A1 (en) * 2008-09-23 2010-03-25 Northwestern University TARGET DNA INTERFERENCE WITH crRNA
EP2362915B1 (fr) 2008-11-07 2016-12-21 DuPont Nutrition Biosciences ApS Séquences crispr de bifidobactéries
PT2367938E (pt) 2008-12-12 2014-09-05 Dupont Nutrition Biosci Aps Agrupamento genético de estirpes de streptococcus thermophilus com propriedades reológicas únicas para a fermentação de lacticínios
WO2010075424A2 (fr) 2008-12-22 2010-07-01 The Regents Of University Of California Compositions et procédés de réduction de gènes procaryotes
GB0823658D0 (en) 2008-12-30 2009-02-04 Angiomed Ag Stent delivery device
US8392349B2 (en) 2009-02-23 2013-03-05 Shalini Vajjhala Global adaptation atlas and method of creating same
EP2419511B1 (fr) 2009-04-09 2018-01-17 Sangamo Therapeutics, Inc. Intégration ciblée dans des cellules souches
DK2424571T3 (da) 2009-04-30 2020-05-04 Ospedale San Raffaele Srl Genvektor
CA2767377A1 (fr) 2009-07-24 2011-01-27 Sigma-Aldrich Co. Llc Procede d'edition de genome
JP5866283B2 (ja) 2009-07-28 2016-02-17 サンガモ バイオサイエンシーズ， インコーポレイテッド トリヌクレオチド反復疾患を治療するための方法および組成物
KR101418355B1 (ko) 2009-10-23 2014-07-11 (주)바이오니아 고밀도 유전자 합성기
DE102009052674B4 (de) 2009-11-12 2012-10-18 Karl Weinhold Verfahren und Vorrichtung zum Verbinden von Doppelmantelrohren
US20110294114A1 (en) 2009-12-04 2011-12-01 Cincinnati Children's Hospital Medical Center Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells
SG181601A1 (en) 2009-12-10 2012-07-30 Univ Minnesota Tal effector-mediated dna modification
EP2534163B1 (fr) 2010-02-09 2015-11-04 Sangamo BioSciences, Inc. Modification génomique ciblée avec des molécules donneuses partiellement monocaténaires
US10087431B2 (en) 2010-03-10 2018-10-02 The Regents Of The University Of California Methods of generating nucleic acid fragments
EP2569425B1 (fr) 2010-05-10 2016-07-06 The Regents of The University of California Compositions d'endoribonucléases et leurs procédés d'utilisation
WO2011146121A1 (fr) 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Nouvelles protéines se liant à l'adn et leurs utilisations
WO2011156430A2 (fr) 2010-06-07 2011-12-15 Fred Hutchinson Cancer Research Center Génération et expression d'endonucléase i-onul génétiquement modifiée et ses homologues et utilisations
EP2392208B1 (fr) * 2010-06-07 2016-05-04 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Protéines de fusion comportant un domaine de liaison ADN d'une protéine TAL effectrice et domaine de clivage non spécifique d'une nucléase de restriction et leur utilisation
EP2580331A4 (fr) 2010-06-14 2013-11-27 Univ Iowa State Res Found Inc Activité nucléase d'un effecteur tal et d'une protéine de fusion foki
WO2012018697A1 (fr) 2010-08-02 2012-02-09 Integrated Dna Technologies, Inc. Procédés pour prédire les températures de stabilité et de fusion de doubles hélices d'acide nucléique
EP2630156B1 (fr) 2010-10-20 2018-08-22 DuPont Nutrition Biosciences ApS Sequences crispr-cas de lactococcus
EP2643343A1 (fr) * 2010-11-26 2013-10-02 Institut Pasteur Identification d'un gyrovirus humain et applications
WO2012087756A1 (fr) 2010-12-22 2012-06-28 Sangamo Biosciences, Inc. Modification par des nucléases à doigts de zinc de fibroblastes mutants de lrrk2 (leucine rich repeat kinase 2) et ipscs
KR20120096395A (ko) 2011-02-22 2012-08-30 주식회사 툴젠 뉴클레아제에 의해 유전자 변형된 세포를 농축시키는 방법
US20140113376A1 (en) 2011-06-01 2014-04-24 Rotem Sorek Compositions and methods for downregulating prokaryotic genes
CN108610423A (zh) 2011-09-21 2018-10-02 桑格摩生物科学股份有限公司 调控转基因表达的方法和组合物
US9458205B2 (en) * 2011-11-16 2016-10-04 Sangamo Biosciences, Inc. Modified DNA-binding proteins and uses thereof
US8450107B1 (en) 2011-11-30 2013-05-28 The Broad Institute Inc. Nucleotide-specific recognition sequences for designer TAL effectors
GB201122458D0 (en) 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
DK2836226T3 (en) 2012-02-24 2017-09-18 Hutchinson Fred Cancer Res COMPOSITIONS AND PROCEDURES FOR TREATING HEMOGLOBINOPATHY
KR102084539B1 (ko) 2012-02-29 2020-03-04 상가모 테라퓨틱스, 인코포레이티드 헌팅턴병을 치료하기 위한 방법 및 조성물
WO2013141680A1 (fr) 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr
AU2013204327B2 (en) 2012-04-20 2016-09-01 Aviagen Cell transfection method
EP2841581B2 (fr) 2012-04-23 2023-03-08 BASF Agricultural Solutions Seed US LLC Modifications ciblées du génome de végétaux
RU2645475C2 (ru) 2012-04-25 2018-02-21 Регенерон Фармасьютикалз, Инк. Опосредованное нуклеазой нацеливание с большими нацеливающими векторами
UA115875C2 (uk) 2012-05-02 2018-01-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Рослина помідора зі зменшеною активністю малатдегідрогенази та спосіб її отримання
KR102116153B1 (ko) 2012-05-07 2020-05-27 상가모 테라퓨틱스, 인코포레이티드 도입유전자의 뉴클레아제-매개 표적화된 통합을 위한 방법 및 조성물
WO2013169398A2 (fr) 2012-05-09 2013-11-14 Georgia Tech Research Corporation Systèmes et procédés pour améliorer la spécificité et l'activité de nucléases
TR201806812T4 (tr) 2012-05-25 2018-06-21 Charpentier Emmanuelle Rna-yönlendirmeli hedef dna modifikasyonu için ve rna-yönlendirmeli transkripsiyon modifikasyonu için yöntemler ve bileşimler.
KR20150027756A (ko) 2012-05-30 2015-03-12 베일러 칼리지 오브 메디신 Dna 수복, 변경 및 대체를 위한 도구로서의 초나선 미니벡터
US9102936B2 (en) 2012-06-11 2015-08-11 Agilent Technologies, Inc. Method of adaptor-dimer subtraction using a CRISPR CAS6 protein
CA2876076A1 (fr) 2012-06-12 2013-12-19 Soren WARMING Procedes et compositions pour la generation d'alleles a inactivation conditionnelle
EP2674501A1 (fr) 2012-06-14 2013-12-18 Agence nationale de sécurité sanitaire de l'alimentation,de l'environnement et du travail Composition de catalyseur pour la polymérisation des oléfines
US9688971B2 (en) 2012-06-15 2017-06-27 The Regents Of The University Of California Endoribonuclease and methods of use thereof
US20150225734A1 (en) 2012-06-19 2015-08-13 Regents Of The University Of Minnesota Gene targeting in plants using dna viruses
EP2872154B1 (fr) 2012-07-11 2017-05-31 Sangamo BioSciences, Inc. Procédés et compositions pour la délivrance d'agents biologiques
EP3196301B1 (fr) 2012-07-11 2018-10-17 Sangamo Therapeutics, Inc. Compositions et procédés pour le traitement de maladies génétiques monofactorielles
EP3808844A1 (fr) 2012-07-25 2021-04-21 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles et outils pour la perturbation du génome et leurs applications
EP2880171B1 (fr) 2012-08-03 2018-10-03 The Regents of The University of California Procédés et compositions permettant de réguler l'expression génique par maturation de l'arn
DK2890780T3 (da) 2012-08-29 2020-09-21 Sangamo Therapeutics Inc Fremgangsmåder og sammensætninger til behandling af en genetisk tilstand
UA119135C2 (uk) 2012-09-07 2019-05-10 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб отримання трансгенної рослини
UA118090C2 (uk) 2012-09-07 2018-11-26 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб інтегрування послідовності нуклеїнової кислоти, що представляє інтерес, у ген fad2 у клітині сої та специфічний для локусу fad2 білок, що зв'язується, здатний індукувати спрямований розрив
BR112015004995B1 (pt) 2012-09-07 2023-05-02 Sangamo Biosciences, Inc. Método para modificação do genoma de uma célula, uso de uma célula, semente ou planta obtida pelo referido método e nuclease de dedo de zinco sítio específica
EP2906602B1 (fr) 2012-10-12 2019-01-16 The General Hospital Corporation Protéines de fusion effecteur de type activateur de transcription (tale) - déméthylase 1 spécifique de la lysine (lsd1)
DE202013012597U1 (de) * 2012-10-23 2017-11-21 Toolgen, Inc. Zusammensetzung zum Spalten einer Ziel-DNA, umfassend eine für die Ziel-DNA spezifische guide-RNA und eine Cas-Protein-codierende Nukleinsäure oder ein Cas-Protein, sowie deren Verwendung
EP2914714A4 (fr) 2012-10-30 2016-09-21 Recombinetics Inc Régulation de la maturation sexuelle chez les animaux
BR112015009812A2 (pt) 2012-10-31 2017-08-22 Cellectis Método para a inserção genética específica em um genoma de planta, célula de planta transformada e seu uso, planta resistente a herbicidas, kit, vetor, e célula hospedeira
US20140127752A1 (en) 2012-11-07 2014-05-08 Zhaohui Zhou Method, composition, and reagent kit for targeted genomic enrichment
KR102145760B1 (ko) 2012-12-06 2020-08-19 시그마-알드리치 컴퍼니., 엘엘씨 Crispr-기초된 유전체 변형과 조절
WO2014093479A1 (fr) 2012-12-11 2014-06-19 Montana State University Contrôle de la régulation génétique guidé par arn crispr (répétitions palindromiques groupées, courtes régulièrement espacées )
SG10201707569YA (en) 2012-12-12 2017-10-30 Broad Inst Inc Delivery, Engineering and Optimization of Systems, Methods and Compositions for Sequence Manipulation and Therapeutic Applications
DK3064585T3 (da) 2012-12-12 2020-04-27 Broad Inst Inc Konstruering og optimering af forbedrede systemer, fremgangsmåder og enzymsammensætninger til sekvensmanipulation
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
ES2553782T3 (es) 2012-12-12 2015-12-11 The Broad Institute, Inc. Ingeniería de sistemas, métodos y composiciones de guía optimizadas para manipulación de secuencias
EP2840140B2 (fr) 2012-12-12 2023-02-22 The Broad Institute, Inc. Procédé utilisant de composants CRISPR-cas pour la mutation de cellules procaryotiques
EP2931899A1 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Génomique fonctionnelle employant des systèmes crispr-cas, des compositions, des procédés, des banques d'inactivation et leurs applications
EP2931892B1 (fr) 2012-12-12 2018-09-12 The Broad Institute, Inc. Procédés, modèles, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
DK2931898T3 (en) * 2012-12-12 2016-06-20 Massachusetts Inst Technology CONSTRUCTION AND OPTIMIZATION OF SYSTEMS, PROCEDURES AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS
US20140310830A1 (en) 2012-12-12 2014-10-16 Feng Zhang CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
EP3031921A1 (fr) 2012-12-12 2016-06-15 The Broad Institute, Inc. Administration, ingénierie et optimisation de systèmes, procédés et compositions pour manipulation de séquence et applications thérapeutiques
KR102240135B1 (ko) 2012-12-13 2021-04-14 다우 아그로사이언시즈 엘엘씨 부위 특이적 뉴클레아제 활성에 대한 dna 검출 방법
CA3081054A1 (fr) 2012-12-17 2014-06-26 President And Fellows Of Harvard College Manipulation du genome humain guidee par l'arn
ES2953523T3 (es) 2012-12-27 2023-11-14 Keygene Nv Método para inducir una translocación dirigida en una planta
AU2014207618A1 (en) 2013-01-16 2015-08-06 Emory University Cas9-nucleic acid complexes and uses related thereto
CN103233028B (zh) 2013-01-25 2015-05-13 南京徇齐生物技术有限公司 一种无物种限制无生物安全性问题的真核生物基因打靶方法及螺旋结构dna序列
WO2014127287A1 (fr) 2013-02-14 2014-08-21 Massachusetts Institute Of Technology Procédé pour la mutagenèse ciblée in vivo
CN105121631B (zh) 2013-02-20 2019-04-19 瑞泽恩制药公司 大鼠的遗传修饰
AU2014218621B2 (en) 2013-02-25 2019-11-07 Sangamo Therapeutics, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
CN111454951A (zh) 2013-03-14 2020-07-28 卡里布生物科学公司 以核酸为靶的核酸的组合物和方法
US20140273235A1 (en) 2013-03-15 2014-09-18 Regents Of The University Of Minnesota ENGINEERING PLANT GENOMES USING CRISPR/Cas SYSTEMS
US20140273230A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
US20140364333A1 (en) 2013-03-15 2014-12-11 President And Fellows Of Harvard College Methods for Live Imaging of Cells
US9234213B2 (en) 2013-03-15 2016-01-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
US10760064B2 (en) 2013-03-15 2020-09-01 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
JP6657069B2 (ja) 2013-03-15 2020-03-04 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
US11332719B2 (en) 2013-03-15 2022-05-17 The Broad Institute, Inc. Recombinant virus and preparations thereof
EP4286517A3 (fr) 2013-04-04 2024-03-13 President and Fellows of Harvard College Utilisations thérapeutiques de l'édition de génome au moyen de systèmes crispr/cas
CA2908512C (fr) 2013-04-05 2023-10-24 Dow Agrosciences Llc Procedes et compositions permettant d'integrer une sequence exogene au sein du genome de plantes
SI2986729T1 (sl) 2013-04-16 2019-02-28 Regeneron Pharmaceuticals, Inc. Ciljana sprememba genoma podgane
CN103224947B (zh) 2013-04-28 2015-06-10 陕西师范大学 一种基因打靶系统
US10604771B2 (en) 2013-05-10 2020-03-31 Sangamo Therapeutics, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
EP2999788B1 (fr) 2013-05-22 2020-07-08 Northwestern University Clivage de l'adn orienté vers arn et édition génétique par l'enzyme cas9 provenant de neisseria meningitidis
US9873907B2 (en) 2013-05-29 2018-01-23 Agilent Technologies, Inc. Method for fragmenting genomic DNA using CAS9
US9267135B2 (en) 2013-06-04 2016-02-23 President And Fellows Of Harvard College RNA-guided transcriptional regulation
EP3597755A1 (fr) 2013-06-17 2020-01-22 The Broad Institute, Inc. Administration, utilisation et applications thérapeutiques des systèmes et compositions crispr-cas pour cibler les troubles et les maladies au moyen de composants viraux
WO2014204727A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Génomique fonctionnelle utilisant des systèmes crispr-cas, procédés de composition, cribles et applications de ces derniers
KR20160019553A (ko) 2013-06-17 2016-02-19 더 브로드 인스티튜트, 인코퍼레이티드 유사분열 후 세포의 질병 및 장애를 표적화하고 모델링하기 위한 시스템, 방법 및 조성물의 전달, 유전자 조작 및 최적화
MX2015017312A (es) 2013-06-17 2017-04-10 Broad Inst Inc Suministro y uso de composiciones, vectores y sistemas crispr-cas para la modificación dirigida y terapia hepáticas.
WO2014204725A1 (fr) 2013-06-17 2014-12-24 The Broad Institute Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
CN103382468B (zh) * 2013-07-04 2015-04-29 中国科学院遗传与发育生物学研究所 一种水稻基因组定点改造方法
CN103343120B (zh) 2013-07-04 2015-03-04 中国科学院遗传与发育生物学研究所 一种小麦基因组定点改造方法
CA3218040A1 (fr) 2013-07-10 2015-01-15 President And Fellows Of Harvard College Proteines cas9 orthogonales pour la regulation et l'edition de genes avec arn guide
CN103388006B (zh) 2013-07-26 2015-10-28 华东师范大学 一种基因定点突变的构建方法
US10421957B2 (en) 2013-07-29 2019-09-24 Agilent Technologies, Inc. DNA assembly using an RNA-programmable nickase
BR102014027466B1 (pt) 2013-11-04 2022-09-27 Dow Agrosciences Llc Molécula de ácido nucleico recombinante, método para produzir uma célula vegetal transgênica e usos de uma planta de soja, parte de planta de soja ou célula de planta de soja transgênica
RU2709734C2 (ru) 2013-11-04 2019-12-19 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Оптимальные локусы маиса
US10233465B2 (en) 2013-11-04 2019-03-19 Dow Agrosciences Llc Optimal soybean loci
AU2014346559B2 (en) 2013-11-07 2020-07-09 Editas Medicine,Inc. CRISPR-related methods and compositions with governing gRNAs
RU2685914C1 (ru) 2013-12-11 2019-04-23 Регенерон Фармасьютикалс, Инк. Способы и композиции для направленной модификации генома
US9850525B2 (en) 2014-01-29 2017-12-26 Agilent Technologies, Inc. CAS9-based isothermal method of detection of specific DNA sequence
WO2015117041A1 (fr) 2014-01-30 2015-08-06 Nair Ramesh B Procédés et compositions à médiation par la modification de gènes pour produire des traits dominants dans les systèmes eucaryotes
US20150225801A1 (en) 2014-02-11 2015-08-13 California Institute Of Technology Recording and mapping lineage information and molecular events in individual cells
US10370680B2 (en) 2014-02-24 2019-08-06 Sangamo Therapeutics, Inc. Method of treating factor IX deficiency using nuclease-mediated targeted integration
US9624498B2 (en) 2014-03-18 2017-04-18 Sangamo Biosciences, Inc. Methods and compositions for regulation of zinc finger protein expression

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055728A1 (en) * 2006-08-04 2010-03-04 Georgia State University Research Foundation, Inc. Enzyme sensors, methods for preparing and using such sensors, and methods of detecting protease activity
US20110217739A1 (en) * 2008-11-06 2011-09-08 University Of Georgia Research Foundation, Inc. Cas6 polypeptides and methods of use
US20120192298A1 (en) * 2009-07-24 2012-07-26 Sigma Aldrich Co. Llc Method for genome editing
WO2012012738A1 (fr) * 2010-07-23 2012-01-26 Sigma-Aldrich Co., Llc Modifications du génome à l'aide d'endonucléases de ciblage et d'acides nucléiques simples brins
WO2013142578A1 (fr) * 2012-03-20 2013-09-26 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-arncr

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GASIUNAS ET AL.: "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.", PNAS, vol. 109, no. 39, 4 September 2012 (2012-09-04), pages E2579 - E2586, XP055068588 *
JINEK ET AL.: "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity", SCIENCE, vol. 337, 17 August 2012 (2012-08-17), pages 816 - 820, XP055229606 *
JINEK ET AL.: "RNA-programmed genome editing in human cells", ELIFE, vol. 2, no. E00471, 29 January 2013 (2013-01-29), pages 1 - 9, XP055245475 *

Cited By (620)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US10982208B2 (en) 2010-11-12 2021-04-20 Gen9, Inc. Protein arrays and methods of using and making the same
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
US11845054B2 (en) 2010-11-12 2023-12-19 Gen9, Inc. Methods and devices for nucleic acids synthesis
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US11566277B2 (en) 2011-12-22 2023-01-31 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11549136B2 (en) 2011-12-22 2023-01-10 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11293052B2 (en) 2011-12-22 2022-04-05 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11293051B2 (en) 2011-12-22 2022-04-05 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11111521B2 (en) 2011-12-22 2021-09-07 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11639518B2 (en) 2011-12-22 2023-05-02 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11566276B2 (en) 2011-12-22 2023-01-31 President And Fellows Of Harvard College Compositions and methods for analyte detection
US11939604B2 (en) 2011-12-30 2024-03-26 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
EP3091072A1 (fr) * 2011-12-30 2016-11-09 Caribou Biosciences, Inc. Ribonucléoprotéines cascade modifiées et utilisations de celles-ci
US10435678B2 (en) 2011-12-30 2019-10-08 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US10954498B2 (en) 2011-12-30 2021-03-23 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
GB2534074A (en) * 2011-12-30 2016-07-13 Caribou Biosciences Inc Modified cascade ribonucleoproteins and uses thereof
GB2512246B (en) * 2011-12-30 2016-07-20 Caribou Biosciences Inc Modified cascade ribonucleoproteins and uses thereof
US10711257B2 (en) 2011-12-30 2020-07-14 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US9885026B2 (en) 2011-12-30 2018-02-06 Caribou Biosciences, Inc. Modified cascade ribonucleoproteins and uses thereof
US9637739B2 (en) 2012-03-20 2017-05-02 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
US10844378B2 (en) 2012-03-20 2020-11-24 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
EP2828386A1 (fr) 2012-03-20 2015-01-28 Vilnius University CLIVAGE D'ADN DIRIGÉ PAR ARN PAR LE COMPLEXE CAS9-ARNcr
EP3594341A1 (fr) * 2012-03-20 2020-01-15 Vilnius University Clivage d'adn dirigé par arn par le complexe cas9-crrna
EP2828386B1 (fr) * 2012-03-20 2019-07-10 Vilnius University CLIVAGE D'ADN DIRIGÉ PAR ARN PAR LE COMPLEXE CAS9-ARNcr
US11555187B2 (en) 2012-03-20 2023-01-17 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
US10927369B2 (en) 2012-04-24 2021-02-23 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US9834786B2 (en) 2012-04-25 2017-12-05 Regeneron Pharmaceuticals, Inc. Nuclease-mediated targeting with large targeting vectors
US10301646B2 (en) 2012-04-25 2019-05-28 Regeneron Pharmaceuticals, Inc. Nuclease-mediated targeting with large targeting vectors
US11898176B2 (en) 2012-04-27 2024-02-13 Duke University Genetic correction of mutated genes
US10550407B2 (en) 2012-05-25 2020-02-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10519467B2 (en) 2012-05-25 2019-12-31 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10308961B2 (en) 2012-05-25 2019-06-04 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10301651B2 (en) 2012-05-25 2019-05-28 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP2800811A1 (fr) 2012-05-25 2014-11-12 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US10337029B2 (en) 2012-05-25 2019-07-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP3241902A1 (fr) * 2012-05-25 2017-11-08 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US11634730B2 (en) 2012-05-25 2023-04-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10266850B2 (en) 2012-05-25 2019-04-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10351878B2 (en) 2012-05-25 2019-07-16 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10358658B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP3401400B1 (fr) 2012-05-25 2019-04-03 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US10358659B2 (en) 2012-05-25 2019-07-23 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10793878B1 (en) 2012-05-25 2020-10-06 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10385360B2 (en) 2012-05-25 2019-08-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11674159B2 (en) 2012-05-25 2023-06-13 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11549127B2 (en) 2012-05-25 2023-01-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10400253B2 (en) 2012-05-25 2019-09-03 The Regents Of The University Of California Methods and compositions or RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11814645B2 (en) 2012-05-25 2023-11-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10407697B2 (en) 2012-05-25 2019-09-10 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11479794B2 (en) 2012-05-25 2022-10-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11473108B2 (en) 2012-05-25 2022-10-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11401532B2 (en) 2012-05-25 2022-08-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10774344B1 (en) 2012-05-25 2020-09-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10415061B2 (en) 2012-05-25 2019-09-17 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10227611B2 (en) 2012-05-25 2019-03-12 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10752920B2 (en) 2012-05-25 2020-08-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982230B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10421980B2 (en) 2012-05-25 2019-09-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10428352B2 (en) 2012-05-25 2019-10-01 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP3241902B1 (fr) 2012-05-25 2018-02-28 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US11332761B2 (en) 2012-05-25 2022-05-17 The Regenis of Wie University of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10982231B2 (en) 2012-05-25 2021-04-20 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988782B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10676759B2 (en) 2012-05-25 2020-06-09 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10443076B2 (en) 2012-05-25 2019-10-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10988780B2 (en) 2012-05-25 2021-04-27 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11001863B2 (en) 2012-05-25 2021-05-11 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10487341B2 (en) 2012-05-25 2019-11-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10669560B2 (en) 2012-05-25 2020-06-02 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11008589B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11293034B2 (en) 2012-05-25 2022-04-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP3401400A1 (fr) * 2012-05-25 2018-11-14 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US10513712B2 (en) 2012-05-25 2019-12-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11274318B2 (en) 2012-05-25 2022-03-15 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10900054B2 (en) 2012-05-25 2021-01-26 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10113167B2 (en) 2012-05-25 2018-10-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
GB2537000A (en) * 2012-05-25 2016-10-05 Univ California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10526619B2 (en) 2012-05-25 2020-01-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10533190B2 (en) 2012-05-25 2020-01-14 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP3597749A1 (fr) * 2012-05-25 2020-01-22 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US11008590B2 (en) 2012-05-25 2021-05-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11242543B2 (en) 2012-05-25 2022-02-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10563227B2 (en) 2012-05-25 2020-02-18 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP2800811B1 (fr) 2012-05-25 2017-05-10 The Regents of The University of California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US11186849B2 (en) 2012-05-25 2021-11-30 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10570419B2 (en) 2012-05-25 2020-02-25 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10000772B2 (en) 2012-05-25 2018-06-19 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11028412B2 (en) 2012-05-25 2021-06-08 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
EP2800811A4 (fr) * 2012-05-25 2015-09-23 Univ California Procédés et compositions permettant la modification de l'adn cible dirigée par l'arn et la modulation de la transcription dirigée par l'arn
US10640791B2 (en) 2012-05-25 2020-05-05 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10577631B2 (en) 2012-05-25 2020-03-03 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
GB2537000B (en) * 2012-05-25 2017-03-08 Univ California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10597680B2 (en) 2012-05-25 2020-03-24 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10612045B2 (en) 2012-05-25 2020-04-07 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US10626419B2 (en) 2012-05-25 2020-04-21 The Regents Of The University Of California Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
US11384360B2 (en) 2012-06-19 2022-07-12 Regents Of The University Of Minnesota Gene targeting in plants using DNA viruses
US11072789B2 (en) 2012-06-25 2021-07-27 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
EP3494997B1 (fr) 2012-07-25 2019-09-18 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles, outils de perturbation du génome et leurs applications
AU2018247306B2 (en) * 2012-07-25 2021-08-19 Massachusetts Institute Of Technology Inducible dna binding proteins and genome perturbation tools and applications thereof
EP3494997A1 (fr) * 2012-07-25 2019-06-12 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles, outils de perturbation du génome et leurs applications
EP3808844A1 (fr) * 2012-07-25 2021-04-21 The Broad Institute, Inc. Protéines de liaison à l'adn inductibles et outils pour la perturbation du génome et leurs applications
JP2016500003A (ja) * 2012-10-23 2016-01-07 ツールゲン インコーポレイテッド 標的ｄｎａに特異的なガイドｒｎａおよびｃａｓタンパク質コード核酸またはｃａｓタンパク質を含む、標的ｄｎａを切断するための組成物、ならびにその使用
US10851380B2 (en) 2012-10-23 2020-12-01 Toolgen Incorporated Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
EP2912175A4 (fr) * 2012-10-23 2015-12-09 Toolgen Inc Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation
JP2016027807A (ja) * 2012-10-23 2016-02-25 ツールゲン インコーポレイテッド 標的ｄｎａに特異的なガイドｒｎａおよびｃａｓタンパク質コード核酸またはｃａｓタンパク質を含む、標的ｄｎａを切断するための組成物、ならびにその使用
AU2017254854B2 (en) * 2012-10-23 2019-12-05 Toolgen Incorporated Composition For Cleaving A Target DNA Comprising A Guide RNA Specific For The Target DNA And Cas Protein-Encoding Nucleic Acid Or Cas Protein, And Use Thereof
EP3346003A1 (fr) * 2012-10-23 2018-07-11 Toolgen Incorporated Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation
EP3733847B1 (fr) 2012-10-23 2022-06-01 Toolgen Incorporated Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation
EP3346003B1 (fr) 2012-10-23 2021-06-09 Toolgen Incorporated Composition pour le clivage d'un adn cible comprenant un arn guide spécifique de l'adn cible et un acide nucléique codant pour la protéine cas ou la protéine cas, et leur utilisation
US10844361B2 (en) 2012-11-16 2020-11-24 Poseida Therapeutics, Inc. Site-specific enzymes and methods of use
US10415024B2 (en) 2012-11-16 2019-09-17 Poseida Therapeutics, Inc. Site-specific enzymes and methods of use
EP3363902B1 (fr) 2012-12-06 2019-11-27 Sigma Aldrich Co. LLC Modification et régulation de génome à base de crispr
US10745716B2 (en) 2012-12-06 2020-08-18 Sigma-Aldrich Co. Llc CRISPR-based genome modification and regulation
US10731181B2 (en) 2012-12-06 2020-08-04 Sigma, Aldrich Co. LLC CRISPR-based genome modification and regulation
EP3138910B1 (fr) 2012-12-06 2017-09-20 Sigma-Aldrich Co. LLC Modification et régulation de génome à base de crispr
EP3360964B1 (fr) 2012-12-06 2019-10-02 Sigma Aldrich Co. LLC Modification et régulation de génome à base de crispr
EP2928496B1 (fr) 2012-12-06 2019-10-09 Sigma Aldrich Co. LLC Modification et régulation du génome basées sur crispr
EP3138912B1 (fr) 2012-12-06 2018-12-05 Sigma Aldrich Co. LLC Modification et régulation de génome à base de crispr
EP3138911B1 (fr) 2012-12-06 2018-12-05 Sigma Aldrich Co. LLC Modification et régulation de génome à base de crispr
JP2022040308A (ja) * 2012-12-12 2022-03-10 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作および治療適用のための系、方法および組成物の送達、エンジニアリングおよび最適化
EP2931898A2 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquence avec des domaines fonctionnels
US10930367B2 (en) 2012-12-12 2021-02-23 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for Cas enzymes or CRISPR-Cas systems for target sequences and conveying results thereof
US9822372B2 (en) 2012-12-12 2017-11-21 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
EP2784162B1 (fr) 2012-12-12 2015-04-08 The Broad Institute, Inc. Ingénierie de systèmes, procédés et compositions de guidage optimisé pour manipulation de séquence
US9840713B2 (en) 2012-12-12 2017-12-12 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8999641B2 (en) 2012-12-12 2015-04-07 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
EP2898075A1 (fr) 2012-12-12 2015-07-29 The Broad Institute, Inc. Fabrication et optimisation de systèmes, procédés et compositions d'enzyme améliorés pour la manipulation de séquences
EP3252160B1 (fr) 2012-12-12 2020-10-28 The Broad Institute, Inc. Systèmes de composants crispr-cas, procédés et compositions pour la manipulation de séquence
US8865406B2 (en) 2012-12-12 2014-10-21 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
JP2016501531A (ja) * 2012-12-12 2016-01-21 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作および治療適用のための系、方法および組成物の送達、エンジニアリングおよび最適化
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
EP2931897B1 (fr) 2012-12-12 2017-11-01 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
JP2016504026A (ja) * 2012-12-12 2016-02-12 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のための系、方法および最適化ガイド組成物のエンジニアリング
EP2764103B1 (fr) 2012-12-12 2015-08-19 The Broad Institute, Inc. Systèmes crispr-cas et procédés pour modifier l'expression de produits de gène
JP2019103513A (ja) * 2012-12-12 2019-06-27 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作および治療適用のための系、方法および組成物の送達、エンジニアリングおよび最適化
JP2019103511A (ja) * 2012-12-12 2019-06-27 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のための系、方法および最適化ガイド組成物のエンジニアリング
US8945839B2 (en) 2012-12-12 2015-02-03 The Broad Institute Inc. CRISPR-Cas systems and methods for altering expression of gene products
US8889418B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
AU2013359262C1 (en) * 2012-12-12 2021-05-13 Massachusetts Institute Of Technology CRISPR-Cas component systems, methods and compositions for sequence manipulation
AU2013359199C1 (en) * 2012-12-12 2021-06-17 Massachusetts Institute Of Technology Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
EP2896697B1 (fr) 2012-12-12 2015-09-02 The Broad Institute, Inc. Ingénierie de systèmes, procédés et compositions de guidage optimisé pour manipulation de séquence
EP2825654B1 (fr) * 2012-12-12 2017-04-26 The Broad Institute, Inc. Systèmes de composants de crispr-cas, procédés et compositions pour la manipulation de séquences
US11041173B2 (en) 2012-12-12 2021-06-22 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
JP2021104055A (ja) * 2012-12-12 2021-07-26 ザ・ブロード・インスティテュート・インコーポレイテッド 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法
EP2931898B1 (fr) 2012-12-12 2016-03-09 The Broad Institute, Inc. Fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquence avec des domaines fonctionnels
AU2017202542B2 (en) * 2012-12-12 2019-07-11 Massachusetts Institute Of Technology Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
JP2021166513A (ja) * 2012-12-12 2021-10-21 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のためのＣＲＩＳＰＲ−Ｃａｓ成分系、方法および組成物
JP2018099131A (ja) * 2012-12-12 2018-06-28 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のための系、方法および最適化ガイド組成物のエンジニアリング
CN106170549B (zh) * 2012-12-12 2020-02-21 布罗德研究所有限公司 用于改变基因产物的表达的crispr-cas系统和方法
AU2019201608B2 (en) * 2012-12-12 2021-12-09 Massachusetts Institute Of Technology CRISPR-Cas systems and methods for altering expression of gene products
JP2019115348A (ja) * 2012-12-12 2019-07-18 ザ・ブロード・インスティテュート・インコーポレイテッ 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法
CN106170549A (zh) * 2012-12-12 2016-11-30 布罗德研究所有限公司 用于改变基因产物的表达的crispr‑cas系统和方法
JP7013406B2 (ja) 2012-12-12 2022-02-15 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作および治療適用のための系、方法および組成物の送達、エンジニアリングおよび最適化
US8932814B2 (en) 2012-12-12 2015-01-13 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8906616B2 (en) 2012-12-12 2014-12-09 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
JP7228059B2 (ja) 2012-12-12 2023-02-22 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作および治療適用のための系、方法および組成物の送達、エンジニアリングおよび最適化
AU2019236591B2 (en) * 2012-12-12 2022-02-24 Massachusetts Institute Of Technology Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
JP2016171817A (ja) * 2012-12-12 2016-09-29 ザ・ブロード・インスティテュート・インコーポレイテッ 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法
EP2998400A1 (fr) * 2012-12-12 2016-03-23 The Broad Institute, Inc. Systèmes et procédés crispr-cas permettant de modifier l'expression de produits géniques
JP2016500262A (ja) * 2012-12-12 2016-01-12 ザ・ブロード・インスティテュート・インコーポレイテッ 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ−Ｃａｓ系および方法
JP7269990B2 (ja) 2012-12-12 2023-05-09 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のためのＣＲＩＳＰＲ－Ｃａｓ成分系、方法および組成物
EP2931897A2 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
JP7090775B2 (ja) 2012-12-12 2022-06-24 ザ・ブロード・インスティテュート・インコーポレイテッド 遺伝子産物の発現を変更するためのＣＲＩＳＰＲ－Ｃａｓ系および方法
EP2764103A2 (fr) 2012-12-12 2014-08-13 The Broad Institute, Inc. Systèmes crispr-cas et procédés pour modifier l'expression de produits de gène
AU2019280394B2 (en) * 2012-12-12 2022-06-09 Massachusetts Institute Of Technology CRISPR-Cas component systems, methods and compositions for sequence manipulation
AU2013359199B2 (en) * 2012-12-12 2019-10-31 Massachusetts Institute Of Technology Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
JP2016129516A (ja) * 2012-12-12 2016-07-21 ザ・ブロード・インスティテュート・インコーポレイテッド 配列操作のための系、方法および最適化ガイド組成物のエンジニアリング
EP2932421A1 (fr) 2012-12-12 2015-10-21 The Broad Institute, Inc. Procédés, systèmes et appareil pour identifier des séquences cibles pour les enzymes cas ou des systèmes crispr-cas pour des séquences cibles et transmettre les résultats associés
AU2013359262B2 (en) * 2012-12-12 2019-10-31 Massachusetts Institute Of Technology CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8889356B2 (en) 2012-12-12 2014-11-18 The Broad Institute Inc. CRISPR-Cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
AU2019283878B2 (en) * 2012-12-12 2022-06-09 Massachusetts Institute Of Technology Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
AU2019229420B2 (en) * 2012-12-12 2022-06-02 Massachusetts Institute Of Technology Engineering of systems, methods and optimized guide compositions for sequence manipulation
EP2921557B1 (fr) 2012-12-12 2016-07-13 The Broad Institute, Inc. Ingénierie de systèmes, procédés et compositions de guidage optimisé pour manipulation de séquence
US8895308B1 (en) 2012-12-12 2014-11-25 The Broad Institute Inc. Engineering and optimization of improved systems, methods and enzyme compositions for sequence manipulation
US10273501B2 (en) 2012-12-17 2019-04-30 President And Fellows Of Harvard College RNA-guided human genome engineering
US11535863B2 (en) 2012-12-17 2022-12-27 President And Fellows Of Harvard College RNA-guided human genome engineering
JP2016501036A (ja) * 2012-12-17 2016-01-18 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性ヒトゲノム改変
EP3553174A1 (fr) * 2012-12-17 2019-10-16 President and Fellows of Harvard College Ingénierie de génome humain guidée par arn
US20140356958A1 (en) * 2012-12-17 2014-12-04 President And Fellows Of Harvard College RNA-Guided Human Genome Engineering
US10717990B2 (en) 2012-12-17 2020-07-21 President And Fellows Of Harvard College RNA-guided human genome engineering
US11359211B2 (en) 2012-12-17 2022-06-14 President And Fellows Of Harvard College RNA-guided human genome engineering
US10435708B2 (en) 2012-12-17 2019-10-08 President And Fellows Of Harvard College RNA-guided human genome engineering
US11365429B2 (en) 2012-12-17 2022-06-21 President And Fellows Of Harvard College RNA-guided human genome engineering
US9023649B2 (en) * 2012-12-17 2015-05-05 President And Fellows Of Harvard College RNA-guided human genome engineering
US11512325B2 (en) 2012-12-17 2022-11-29 President And Fellows Of Harvard College RNA-guided human genome engineering
AU2013363194B2 (en) * 2012-12-17 2019-05-16 President And Fellows Of Harvard College RNA-guided human genome engineering
EP4282970A3 (fr) * 2012-12-17 2024-01-17 President and Fellows of Harvard College Ingénierie de génome humain guidée par arn
JP2021048882A (ja) * 2012-12-17 2021-04-01 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性ヒトゲノム改変
US9260723B2 (en) 2012-12-17 2016-02-16 President And Fellows Of Harvard College RNA-guided human genome engineering
US11236359B2 (en) 2012-12-17 2022-02-01 President And Fellows Of Harvard College RNA-guided human genome engineering
EP2931891A4 (fr) * 2012-12-17 2016-07-13 Harvard College Manipulation du génome humain guidée par l'arn
US9970024B2 (en) 2012-12-17 2018-05-15 President And Fellows Of Harvard College RNA-guided human genome engineering
EP2922393A1 (fr) 2013-02-27 2015-09-30 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Édition de gène dans l'ovocyte au moyen de cas9 nucléases
JP2016507244A (ja) * 2013-02-27 2016-03-10 ヘルムホルツ・ツェントルム・ミュンヒェン・ドイチェス・フォルシュンクスツェントルム・フューア・ゲズントハイト・ウント・ウムベルト（ゲーエムベーハー）Ｈｅｌｍｈｏｌｔｚ Ｚｅｎｔｒｕｍ ＭｕｅｎｃｈｅｎＤｅｕｔｓｃｈｅｓ Ｆｏｒｓｃｈｕｎｇｓｚｅｎｔｒｕｍ ｆｕｅｒ Ｇｅｓｕｎｄｈｅｉｔ ｕｎｄ Ｕｍｗｅｌｔ （ＧｍｂＨ） Ｃａｓ９ヌクレアーゼによる卵母細胞における遺伝子編集
US9783780B2 (en) 2013-02-27 2017-10-10 Helmholtz Zentrum München Deutsches Forschungszentrum Für Gesundheit Und Umwelt Gene editing in the oocyte by CAS9 nucleases
US10214723B2 (en) 2013-02-27 2019-02-26 Helmholtz Zentrum München Deutsches Forschungszentrum Für Gesundheit Und Umwelt Gene editing in the oocyte by Cas9 nucleases
US11299767B2 (en) 2013-03-12 2022-04-12 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
US9809814B1 (en) 2013-03-14 2017-11-07 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9909122B2 (en) 2013-03-14 2018-03-06 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9260752B1 (en) 2013-03-14 2016-02-16 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9725714B2 (en) 2013-03-14 2017-08-08 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US11312953B2 (en) 2013-03-14 2022-04-26 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9803194B2 (en) 2013-03-14 2017-10-31 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
US9410198B2 (en) 2013-03-14 2016-08-09 Caribou Biosciences, Inc. Compostions and methods of nucleic acid-targeting nucleic acids
US10125361B2 (en) 2013-03-14 2018-11-13 Caribou Biosciences, Inc. Compositions and methods of nucleic acid-targeting nucleic acids
JP7053706B2 (ja) 2013-03-15 2022-04-12 ザ ジェネラル ホスピタル コーポレイション 短縮ガイドＲＮＡ（ｔｒｕ－ｇＲＮＡ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US10415059B2 (en) 2013-03-15 2019-09-17 The General Hospital Corporation Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
US9567603B2 (en) 2013-03-15 2017-02-14 The General Hospital Corporation Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing
JP2021118726A (ja) * 2013-03-15 2021-08-12 ザ ジェネラル ホスピタル コーポレイション ＲＮＡ誘導型ＦｏｋＩヌクレアーゼ（ＲＦＮ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US9567604B2 (en) 2013-03-15 2017-02-14 The General Hospital Corporation Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
JP2020031637A (ja) * 2013-03-15 2020-03-05 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
US11098326B2 (en) 2013-03-15 2021-08-24 The General Hospital Corporation Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing
JP2016512264A (ja) * 2013-03-15 2016-04-25 ザ ジェネラル ホスピタル コーポレイション 特定のゲノム遺伝子座へのゲノムおよびエピゲノム調節タンパク質のｒｎａ誘導型標的化
US11920152B2 (en) 2013-03-15 2024-03-05 The General Hospital Corporation Increasing specificity for RNA-guided genome editing
JP2016512048A (ja) * 2013-03-15 2016-04-25 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ ＣＲＩＳＰＲ／Ｃａｓシステムを使用した植物ゲノム操作
JP2020039350A (ja) * 2013-03-15 2020-03-19 ザ ジェネラル ホスピタル コーポレイション ＲＮＡ誘導型ＦｏｋＩヌクレアーゼ（ＲＦＮ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US10544433B2 (en) 2013-03-15 2020-01-28 The General Hospital Corporation Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing
US11168338B2 (en) 2013-03-15 2021-11-09 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
JP2016512691A (ja) * 2013-03-15 2016-05-09 ザ ジェネラル ホスピタル コーポレイション 短縮ガイドＲＮＡ（ｔｒｕ−ｇＲＮＡ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
JP7126588B2 (ja) 2013-03-15 2022-08-26 ザ ジェネラル ホスピタル コーポレイション ＲＮＡ誘導型ＦｏｋＩヌクレアーゼ（ＲＦＮ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US10526589B2 (en) 2013-03-15 2020-01-07 The General Hospital Corporation Multiplex guide RNAs
US10119133B2 (en) 2013-03-15 2018-11-06 The General Hospital Corporation Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
JP2019205470A (ja) * 2013-03-15 2019-12-05 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ ＣＲＩＳＰＲ／Ｃａｓシステムを使用した植物ゲノム操作
US10138476B2 (en) 2013-03-15 2018-11-27 The General Hospital Corporation Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing
US10378027B2 (en) 2013-03-15 2019-08-13 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
JP2020120661A (ja) * 2013-03-15 2020-08-13 ザ ジェネラル ホスピタル コーポレイション 短縮ガイドＲＮＡ（ｔｒｕ−ｇＲＮＡ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US11634731B2 (en) 2013-03-15 2023-04-25 The General Hospital Corporation Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing
US9885033B2 (en) 2013-03-15 2018-02-06 The General Hospital Corporation Increasing specificity for RNA-guided genome editing
US10760064B2 (en) 2013-03-15 2020-09-01 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
JP2016517276A (ja) * 2013-03-15 2016-06-16 ザ ジェネラル ホスピタル コーポレイション ＲＮＡ誘導型ＦｏｋＩヌクレアーゼ（ＲＦＮ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US10844403B2 (en) 2013-03-15 2020-11-24 The General Hospital Corporation Increasing specificity for RNA-guided genome editing
JP7379572B2 (ja) 2013-03-15 2023-11-14 ザ ジェネラル ホスピタル コーポレイション 短縮ガイドＲＮＡ（ｔｒｕ－ｇＲＮＡ）を用いたＲＮＡ誘導型ゲノム編集の特異性の増大
US20160186208A1 (en) * 2013-04-16 2016-06-30 Whitehead Institute For Biomedical Research Methods of Mutating, Modifying or Modulating Nucleic Acid in a Cell or Nonhuman Mammal
US10975390B2 (en) 2013-04-16 2021-04-13 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
US10385359B2 (en) 2013-04-16 2019-08-20 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
US10196652B2 (en) 2013-05-15 2019-02-05 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
JP2016521975A (ja) * 2013-05-15 2016-07-28 サンガモ バイオサイエンシーズ， インコーポレイテッド 遺伝的状態の処置のための方法および組成物
US9902974B2 (en) * 2013-05-15 2018-02-27 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
US20160090607A1 (en) * 2013-05-15 2016-03-31 Sangamo Biosciences, Inc. Methods and compositions for treatment of a genetic condition
US10196651B2 (en) 2013-05-15 2019-02-05 Sangamo Therapeutics, Inc. Methods and compositions for treatment of a genetic condition
JP2021003107A (ja) * 2013-05-29 2021-01-14 セレクティスＣｅｌｌｅｃｔｉｓ Ｃａｓ９ニッカーゼ活性を用いて正確なｄｎａ切断をもたらすための方法
EP3004349A1 (fr) * 2013-05-29 2016-04-13 Cellectis S.A. Procédé visant à produire un clivage d'adn précis par l'activité de cas9 nickase
EP3004349B1 (fr) * 2013-05-29 2018-03-28 Cellectis S.A. Procédé visant à produire un clivage d'adn précis par l'activité de cas9 nickase
JP2016520317A (ja) * 2013-05-29 2016-07-14 セレクティスＣｅｌｌｅｃｔｉｓ Ｃａｓ９ニッカーゼ活性を用いて正確なｄｎａ切断をもたらすための方法
JP7036511B2 (ja) 2013-06-04 2022-03-15 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
EP3003392A4 (fr) * 2013-06-04 2017-05-03 President and Fellows of Harvard College Régulation de la transcription à guidage arn
JP2022166077A (ja) * 2013-06-04 2022-11-01 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
AU2020203977B2 (en) * 2013-06-04 2021-05-27 President And Fellows Of Harvard College RNA-guided transcriptional regulation
US10767194B2 (en) 2013-06-04 2020-09-08 President And Fellows Of Harvard College RNA-guided transcriptional regulation
EP3603679A1 (fr) * 2013-06-04 2020-02-05 President and Fellows of Harvard College Régulation transcriptionnelle guidée par arn
US10640789B2 (en) 2013-06-04 2020-05-05 President And Fellows Of Harvard College RNA-guided transcriptional regulation
JP2019122384A (ja) * 2013-06-04 2019-07-25 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
JP7376650B2 (ja) 2013-06-04 2023-11-08 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
JP7119055B2 (ja) 2013-06-04 2022-08-16 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
JP2016521554A (ja) * 2013-06-04 2016-07-25 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
AU2014274939B2 (en) * 2013-06-04 2020-03-19 President And Fellows Of Harvard College RNA-guideded transcriptional regulation
JP2021045153A (ja) * 2013-06-04 2021-03-25 プレジデント アンド フェローズ オブ ハーバード カレッジ Ｒｎａ誘導性転写制御
EP4159243A1 (fr) * 2013-06-04 2023-04-05 President and Fellows of Harvard College Régulation transcriptionnelle guidée par arn
EP3008186B1 (fr) 2013-06-14 2018-11-28 Cellectis Procédés d'édition de génome non transgénique dans des plantes
US20160186213A1 (en) * 2013-06-17 2016-06-30 The Broad Institute Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US11008588B2 (en) 2013-06-17 2021-05-18 The Broad Institute, Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US11597949B2 (en) 2013-06-17 2023-03-07 The Broad Institute, Inc. Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
EP3011030B1 (fr) * 2013-06-17 2023-11-08 The Broad Institute, Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
US10711285B2 (en) 2013-06-17 2020-07-14 The Broad Institute, Inc. Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
JP2016521995A (ja) * 2013-06-17 2016-07-28 ザ・ブロード・インスティテュート・インコーポレイテッド ウイルス成分を使用して障害および疾患をターゲティングするためのＣＲＩＳＰＲ−Ｃａｓ系および組成物の送達、使用および治療上の適用
US10577630B2 (en) 2013-06-17 2020-03-03 The Broad Institute, Inc. Delivery and use of the CRISPR-Cas systems, vectors and compositions for hepatic targeting and therapy
US10946108B2 (en) 2013-06-17 2021-03-16 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for targeting disorders and diseases using viral components
JP2016524472A (ja) * 2013-06-17 2016-08-18 ザ・ブロード・インスティテュート・インコーポレイテッド 肝臓ターゲティングおよび治療のためのＣＲＩＳＰＲ−Ｃａｓ系、ベクターおよび組成物の送達および使用
EP3011030A1 (fr) * 2013-06-17 2016-04-27 The Broad Institute, Inc. Systèmes, procédés et compositions à double nickase crispr-cas optimisés, pour la manipulation de séquences
EP3597755A1 (fr) * 2013-06-17 2020-01-22 The Broad Institute, Inc. Administration, utilisation et applications thérapeutiques des systèmes et compositions crispr-cas pour cibler les troubles et les maladies au moyen de composants viraux
US10781444B2 (en) 2013-06-17 2020-09-22 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
JP2016523082A (ja) * 2013-06-17 2016-08-08 ザ・ブロード・インスティテュート・インコーポレイテッド 分裂終了細胞の疾患および障害をターゲティングおよびモデリングするための系、方法および組成物の送達、エンジニアリングおよび最適化
JP2020063238A (ja) * 2013-06-17 2020-04-23 ザ・ブロード・インスティテュート・インコーポレイテッド 分裂終了細胞の疾患および障害をターゲティングおよびモデリングするための系、方法および組成物の送達、エンジニアリングおよび最適化
US10011850B2 (en) 2013-06-21 2018-07-03 The General Hospital Corporation Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
JP2016528894A (ja) * 2013-07-26 2016-09-23 プレジデント アンド フェローズ オブ ハーバード カレッジ ゲノムエンジニアリング
US11306328B2 (en) 2013-07-26 2022-04-19 President And Fellows Of Harvard College Genome engineering
US10563225B2 (en) 2013-07-26 2020-02-18 President And Fellows Of Harvard College Genome engineering
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US9163284B2 (en) 2013-08-09 2015-10-20 President And Fellows Of Harvard College Methods for identifying a target site of a Cas9 nuclease
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US11773400B2 (en) 2013-08-22 2023-10-03 E.I. Du Pont De Nemours And Company Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof
US10519457B2 (en) 2013-08-22 2019-12-31 E I Du Pont De Nemours And Company Soybean U6 polymerase III promoter and methods of use
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
EP3988649A1 (fr) * 2013-09-18 2022-04-27 Kymab Limited Procédés, cellules et organismes
US11920128B2 (en) 2013-09-18 2024-03-05 Kymab Limited Methods, cells and organisms
US11149267B2 (en) 2013-10-28 2021-10-19 The Broad Institute, Inc. Functional genomics using CRISPR-Cas systems, compositions, methods, screens and applications thereof
US10640788B2 (en) 2013-11-07 2020-05-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAs
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US10190137B2 (en) 2013-11-07 2019-01-29 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US11390887B2 (en) 2013-11-07 2022-07-19 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US10208317B2 (en) 2013-12-11 2019-02-19 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse embryonic stem cell genome
US11820997B2 (en) 2013-12-11 2023-11-21 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a genome
US10711280B2 (en) 2013-12-11 2020-07-14 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse ES cell genome
EP3460063A1 (fr) 2013-12-11 2019-03-27 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
US9546384B2 (en) 2013-12-11 2017-01-17 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a mouse genome
WO2015088643A1 (fr) 2013-12-11 2015-06-18 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
US9228208B2 (en) 2013-12-11 2016-01-05 Regeneron Pharmaceuticals, Inc. Methods and compositions for the targeted modification of a genome
EP4349980A2 (fr) 2013-12-11 2024-04-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification ciblée d'un génome
US9068179B1 (en) 2013-12-12 2015-06-30 President And Fellows Of Harvard College Methods for correcting presenilin point mutations
US11155795B2 (en) 2013-12-12 2021-10-26 The Broad Institute, Inc. CRISPR-Cas systems, crystal structure and uses thereof
AU2014362208B2 (en) * 2013-12-12 2021-02-11 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11597919B2 (en) 2013-12-12 2023-03-07 The Broad Institute Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
AU2021200375B2 (en) * 2013-12-12 2023-08-17 President And Fellows Of Harvard College Cas variants for gene editing
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11407985B2 (en) 2013-12-12 2022-08-09 The Broad Institute, Inc. Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for genome editing
US10377998B2 (en) 2013-12-12 2019-08-13 The Broad Institute, Inc. CRISPR-CAS systems and methods for altering expression of gene products, structural information and inducible modular CAS enzymes
US10550372B2 (en) 2013-12-12 2020-02-04 The Broad Institute, Inc. Systems, methods and compositions for sequence manipulation with optimized functional CRISPR-Cas systems
US11149259B2 (en) 2013-12-12 2021-10-19 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products, structural information and inducible modular Cas enzymes
US10851357B2 (en) 2013-12-12 2020-12-01 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US11591581B2 (en) 2013-12-12 2023-02-28 The Broad Institute, Inc. Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders
US11028388B2 (en) 2014-03-05 2021-06-08 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa
US9938521B2 (en) 2014-03-10 2018-04-10 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating leber's congenital amaurosis 10 (LCA10)
US11141493B2 (en) 2014-03-10 2021-10-12 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
US10253312B2 (en) 2014-03-10 2019-04-09 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating Leber's Congenital Amaurosis 10 (LCA10)
US11268086B2 (en) 2014-03-10 2022-03-08 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating Leber's Congenital Amaurosis 10 (LCA10)
US11339437B2 (en) 2014-03-10 2022-05-24 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
US20170007679A1 (en) * 2014-03-25 2017-01-12 Editas Medicine Inc. Crispr/cas-related methods and compositions for treating hiv infection and aids
US11242525B2 (en) 2014-03-26 2022-02-08 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating sickle cell disease
US9970001B2 (en) 2014-06-05 2018-05-15 Sangamo Therapeutics, Inc. Methods and compositions for nuclease design
EP3152319A4 (fr) * 2014-06-05 2017-12-27 Sangamo BioSciences, Inc. Méthodes et compositions pour conception de nucléases
EP3708671A1 (fr) 2014-06-06 2020-09-16 Regeneron Pharmaceuticals, Inc. Procédés et compositions permettant la modification d'un locus cible
WO2015188109A1 (fr) 2014-06-06 2015-12-10 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour la modification d'un locus ciblé
US10406245B2 (en) 2014-06-16 2019-09-10 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter
EA039693B1 (ru) * 2014-06-16 2022-02-28 Дзе Джонс Хопкинс Юниверсити Композиции и способы для экспрессии рнк-проводников crispr с использованием промотора h1
US11896679B2 (en) 2014-06-16 2024-02-13 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter
CN106852157A (zh) * 2014-06-16 2017-06-13 约翰斯·霍普金斯大学 用于使用h1启动子表达crispr向导rna的组合物和方法
US10668173B2 (en) * 2014-06-16 2020-06-02 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAS using the H1 promoter
US10369234B2 (en) 2014-06-16 2019-08-06 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter
US9907863B2 (en) 2014-06-16 2018-03-06 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter
JP2017519500A (ja) * 2014-06-16 2017-07-20 ザ・ジョンズ・ホプキンス・ユニバーシティー Ｈ１プロモーターを用いるｃｒｉｓｐｒガイドｒｎａの発現のための組成物および方法
JP2021052809A (ja) * 2014-06-16 2021-04-08 ザ・ジョンズ・ホプキンス・ユニバーシティー Ｈ１プロモーターを用いるｃｒｉｓｐｒガイドｒｎａの発現のための組成物および方法
AU2015277369B2 (en) * 2014-06-16 2021-08-19 The Johns Hopkins University Compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter
WO2015195621A1 (fr) * 2014-06-16 2015-12-23 The Johns Hopkins University Compositions et procédés pour l'expression d'arn guide de crispr à l'aide du promoteur h1
JP2017518082A (ja) * 2014-06-17 2017-07-06 ポセイダ セラピューティクス， インコーポレイテッド ゲノム中の特異的遺伝子座にタンパク質を指向させるための方法およびその使用
US9738897B2 (en) 2014-06-23 2017-08-22 Regeneron Pharmaceuticals, Inc. Nuclease-mediated DNA assembly
US10626402B2 (en) 2014-06-23 2020-04-21 Regeneron Pharmaceuticals, Inc. Nuclease-mediated DNA assembly
EP3354732A1 (fr) 2014-06-23 2018-08-01 Regeneron Pharmaceuticals, Inc. Assemblage d'adn mediée par nuclease
US9580715B2 (en) 2014-06-23 2017-02-28 Regeneron Pharmaceuticals, Inc. Nuclease-mediated DNA assembly
US11932859B2 (en) 2014-06-23 2024-03-19 Regeneron Pharmaceuticals, Inc. Nuclease-mediated DNA assembly
US10273488B2 (en) 2014-06-23 2019-04-30 Regeneron Pharmaceuticals, Inc. Nuclease-mediated DNA assembly
WO2015200334A1 (fr) * 2014-06-23 2015-12-30 Regeneron Pharmaceuticals, Inc. Assemblage d'adn à médiation par une nucléase
EP3708663A1 (fr) 2014-06-23 2020-09-16 Regeneron Pharmaceuticals, Inc. Assemblage d'adn mediée par nuclease
CN106715694A (zh) * 2014-06-23 2017-05-24 瑞泽恩制药公司 核酸酶介导的dna组装
WO2015200805A2 (fr) 2014-06-26 2015-12-30 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétiques ciblées et procédés d'utilisation
EP3461885A1 (fr) 2014-06-26 2019-04-03 Regeneron Pharmaceuticals, Inc. Procèdes et compositions pour la modification génétique ciblé et procèdes d'utilisation
US20170198268A1 (en) * 2014-07-09 2017-07-13 Gen9, Inc. Compositions and Methods for Site-Directed DNA Nicking and Cleaving
WO2016011080A3 (fr) * 2014-07-14 2016-03-03 The Regents Of The University Of California Modulation transcriptionnelle par crispr/cas
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10519454B2 (en) 2014-08-06 2019-12-31 Toolgen Incorporated Genome editing using Campylobacter jejuni CRISPR/CAS system-derived RGEN
JP2017526387A (ja) * 2014-08-06 2017-09-14 トゥールジェン インコーポレイテッド カンピロバクター・ジェジュニｃｒｉｓｐｒ／ｃａｓシステムに由来するｒｇｅｎを使用したゲノム編集
WO2016036754A1 (fr) 2014-09-02 2016-03-10 The Regents Of The University Of California Procédés et compositions pour assurer la modification de l'adn cible arn dépendante
US11560568B2 (en) 2014-09-12 2023-01-24 E. I. Du Pont De Nemours And Company Generation of site-specific-integration sites for complex trait loci in corn and soybean, and methods of use
EP3204399A4 (fr) * 2014-10-09 2018-04-25 Seattle Children's Hospital, dba Seattle Children's Research Institute Longs plasmides poly (a) et méthodes d'introduction de longues séquences poly(a) dans le plasmide
US11400169B2 (en) 2014-10-09 2022-08-02 Seattle Children's Hospital Long poly(A) plasmids and methods for introduction of long poly(A) sequences into the plasmid
EP3998344A1 (fr) * 2014-10-09 2022-05-18 Life Technologies Corporation Oligonucléotides crispr et édition de gènes
EP3561052A1 (fr) 2014-10-15 2019-10-30 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
WO2016061374A1 (fr) 2014-10-15 2016-04-21 Regeneron Pharmaceuticals, Inc. Méthodes et compositions de production ou de conservation de cellules pluripotentes
US20230151345A1 (en) * 2014-10-24 2023-05-18 Life Technologies Corporation Compositions and methods for enhancing homologous recombination
CN107429246A (zh) * 2014-10-31 2017-12-01 麻省理工学院 用于crispr的大规模并行组合遗传学
US10208298B2 (en) 2014-11-06 2019-02-19 E.I. Du Pont De Nemours And Company Peptide-mediated delivery of RNA-guided endonuclease into cells
US10584322B2 (en) 2014-11-06 2020-03-10 Dupont Us Holding, Llc Peptide-mediated delivery of RNA-guided endonuclease into cells
EA038321B1 (ru) * 2014-11-06 2021-08-09 Е.И. Дюпон Де Немур Энд Компани Опосредуемая пептидом доставка направляемой рнк эндонуклеазы в клетки
WO2016073433A1 (fr) * 2014-11-06 2016-05-12 E. I. Du Pont De Nemours And Company Administration médiée par un peptide d'endonucléase guidée par un arn dans des cellules
JP2017532979A (ja) * 2014-11-06 2017-11-09 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニーＥ．Ｉ．Ｄｕ Ｐｏｎｔ Ｄｅ Ｎｅｍｏｕｒｓ Ａｎｄ Ｃｏｍｐａｎｙ Ｒｎａ誘導型エンドヌクレアーゼの細胞へのペプチド媒介輸送
EP3597740A1 (fr) * 2014-11-06 2020-01-22 E. I. du Pont de Nemours and Company Administration à médiation de peptides d'endonucléases guidées par arn dans des cellules
AU2015343307B2 (en) * 2014-11-06 2021-05-20 Iff Us Holding, Llc Peptide-mediated delivery of RNA-guided endonuclease into cells
CN107406838A (zh) * 2014-11-06 2017-11-28 纳幕尔杜邦公司 Rna引导的内切核酸酶向细胞中的肽介导的递送
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US10858662B2 (en) 2014-11-19 2020-12-08 Institute For Basic Science Genome editing with split Cas9 expressed from two vectors
WO2016080795A1 (fr) * 2014-11-19 2016-05-26 기초과학연구원 Procédé de régulation de l'expression génique à l'aide de la protéine cas9 exprimée par deux vecteurs
EP3521437A1 (fr) 2014-11-21 2019-08-07 Regeneron Pharmaceuticals, Inc. Methodes et compositions pour la modification génétique ciblée en utilisant des arn de guidage assemblés par paires
WO2016081923A2 (fr) 2014-11-21 2016-05-26 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée utilisant des arn guides appariés
WO2016083811A1 (fr) 2014-11-27 2016-06-02 Imperial Innovations Limited Procédés d'édition de génome
US10900034B2 (en) 2014-12-03 2021-01-26 Agilent Technologies, Inc. Guide RNA with chemical modifications
US10337001B2 (en) 2014-12-03 2019-07-02 Agilent Technologies, Inc. Guide RNA with chemical modifications
US11234418B2 (en) 2014-12-10 2022-02-01 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10993419B2 (en) 2014-12-10 2021-05-04 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10278372B2 (en) 2014-12-10 2019-05-07 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10696986B2 (en) 2014-12-12 2020-06-30 The Board Institute, Inc. Protected guide RNAS (PGRNAS)
US11624078B2 (en) 2014-12-12 2023-04-11 The Broad Institute, Inc. Protected guide RNAS (pgRNAS)
WO2016100857A1 (fr) 2014-12-19 2016-06-23 Regeneron Pharmaceuticals, Inc. Cellules souches comme modèle du diabète de type 2
EP3653048A1 (fr) 2014-12-19 2020-05-20 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour modification génétique ciblée par ciblage multiple en une seule étape
EP3798302A1 (fr) 2015-01-29 2021-03-31 Meiogenix Procede pour induire des recombinaisons meiotiques ciblees
WO2016120480A1 (fr) 2015-01-29 2016-08-04 Meiogenix Procede pour induire des recombinaisons meiotiques ciblees
US11535846B2 (en) 2015-04-06 2022-12-27 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAS for CRISPR/Cas-mediated gene regulation
US11851652B2 (en) 2015-04-06 2023-12-26 The Board Of Trustees Of The Leland Stanford Junior Compositions comprising chemically modified guide RNAs for CRISPR/Cas-mediated editing of HBB
US11306309B2 (en) 2015-04-06 2022-04-19 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation
US11180793B2 (en) 2015-04-24 2021-11-23 Editas Medicine, Inc. Evaluation of Cas9 molecule/guide RNA molecule complexes
WO2016176617A2 (fr) 2015-04-29 2016-11-03 New York University Procédé pour le traitement de gliomes de haut grade
WO2016178207A1 (fr) 2015-05-04 2016-11-10 Ramot At Tel-Aviv University Ltd. Procédés et kits pour fragmenter l'adn
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US11911415B2 (en) 2015-06-09 2024-02-27 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for improving transplantation
JP2021112211A (ja) * 2015-06-17 2021-08-05 ポセイダ セラピューティクス， インコーポレイテッド タンパク質をゲノム内の特定の遺伝子座に導くための組成物および方法
CN108026519A (zh) * 2015-06-17 2018-05-11 波赛达治疗公司 将蛋白质定向至基因组中的特定基因座的组合物和方法
JP2018521689A (ja) * 2015-06-17 2018-08-09 ポセイダ セラピューティクス， インコーポレイテッド タンパク質をゲノム内の特定の遺伝子座に導くための組成物および方法
JP7170090B2 (ja) 2015-06-17 2022-11-11 ポセイダ セラピューティクス， インコーポレイテッド タンパク質をゲノム内の特定の遺伝子座に導くための組成物および方法
US11473082B2 (en) 2015-06-17 2022-10-18 Poseida Therapeutics, Inc. Compositions and methods for directing proteins to specific loci in the genome
AU2021250992B2 (en) * 2015-06-17 2023-09-28 Poseida Therapeutics, Inc. Compositions and methods for directing proteins to specific loci in the genome
AU2016278226B2 (en) * 2015-06-17 2021-08-12 Poseida Therapeutics, Inc. Compositions and methods for directing proteins to specific loci in the genome
WO2016205554A1 (fr) * 2015-06-17 2016-12-22 Poseida Therapeutics, Inc. Compositions et procédés permettant de diriger des protéines vers des loci spécifiques dans le génome
EP3929286A1 (fr) * 2015-06-17 2021-12-29 Poseida Therapeutics, Inc. Compositions et procédés permettant de diriger des protéines vers des loci spécifiques dans le génome
US11578312B2 (en) 2015-06-18 2023-02-14 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of CAS9 orthologs and variants for sequence manipulation
US10494621B2 (en) 2015-06-18 2019-12-03 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
US10876100B2 (en) 2015-06-18 2020-12-29 The Broad Institute, Inc. Crispr enzyme mutations reducing off-target effects
WO2016210271A1 (fr) * 2015-06-24 2016-12-29 Sigma-Aldrich Co. Llc Modification et régulation génomique dépendantes du cycle cellulaire
US10166255B2 (en) 2015-07-31 2019-01-01 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11903966B2 (en) 2015-07-31 2024-02-20 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11642374B2 (en) 2015-07-31 2023-05-09 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
US11642375B2 (en) 2015-07-31 2023-05-09 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
US11147837B2 (en) 2015-07-31 2021-10-19 Regents Of The University Of Minnesota Modified cells and methods of therapy
US11925664B2 (en) 2015-07-31 2024-03-12 Intima Bioscience, Inc. Intracellular genomic transplant and methods of therapy
US10406177B2 (en) 2015-07-31 2019-09-10 Regents Of The University Of Minnesota Modified cells and methods of therapy
US11266692B2 (en) 2015-07-31 2022-03-08 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
US11583556B2 (en) 2015-07-31 2023-02-21 Regents Of The University Of Minnesota Modified cells and methods of therapy
WO2017027910A1 (fr) 2015-08-14 2017-02-23 The University Of Sydney Inhibition de la connexine 45 pour la thérapie
EP4043074A1 (fr) 2015-08-14 2022-08-17 The University of Sydney Inhibition de la connexine 45 pour la thérapie
US11427817B2 (en) 2015-08-25 2022-08-30 Duke University Compositions and methods of improving specificity in genomic engineering using RNA-guided endonucleases
US10633642B2 (en) 2015-08-28 2020-04-28 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
US10526591B2 (en) 2015-08-28 2020-01-07 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
WO2017040348A1 (fr) 2015-08-28 2017-03-09 The General Hospital Corporation Nucléases crispr-cas9 modifiées
US9926546B2 (en) 2015-08-28 2018-03-27 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
US10093910B2 (en) 2015-08-28 2018-10-09 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
US11060078B2 (en) 2015-08-28 2021-07-13 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
EP4036236A1 (fr) 2015-08-28 2022-08-03 The General Hospital Corporation Nucléases crispr-cas9 modifiées
US9512446B1 (en) 2015-08-28 2016-12-06 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
US11667911B2 (en) 2015-09-24 2023-06-06 Editas Medicine, Inc. Use of exonucleases to improve CRISPR/CAS-mediated genome editing
EP4089175A1 (fr) * 2015-10-13 2022-11-16 Duke University Ingénierie génomique avec systèmes crispr de type i dans des cellules eucaryotes
EP3362571A4 (fr) * 2015-10-13 2019-07-10 Duke University Ingénierie génomique avec systèmes crispr de type i dans des cellules eucaryotes
US11421251B2 (en) 2015-10-13 2022-08-23 Duke University Genome engineering with type I CRISPR systems in eukaryotic cells
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US20170121693A1 (en) * 2015-10-23 2017-05-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
WO2017075335A1 (fr) 2015-10-28 2017-05-04 Voyager Therapeutics, Inc. Expression régulable au moyen d'un virus adéno-associé (vaa)
US11542554B2 (en) 2015-11-03 2023-01-03 President And Fellows Of Harvard College Method and apparatus for volumetric imaging
US10240145B2 (en) * 2015-11-25 2019-03-26 The Board Of Trustees Of The Leland Stanford Junior University CRISPR/Cas-mediated genome editing to treat EGFR-mutant lung cancer
WO2017158153A1 (fr) 2016-03-17 2017-09-21 Imba - Institut Für Molekulare Biotechnologie Gmbh Expression conditionnelle de sgrna de crispr
EP3219799A1 (fr) 2016-03-17 2017-09-20 IMBA-Institut für Molekulare Biotechnologie GmbH Expression sgrna crispr conditionnelle
US11597924B2 (en) 2016-03-25 2023-03-07 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US11512311B2 (en) 2016-03-25 2022-11-29 Editas Medicine, Inc. Systems and methods for treating alpha 1-antitrypsin (A1AT) deficiency
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US11713485B2 (en) 2016-04-25 2023-08-01 President And Fellows Of Harvard College Hybridization chain reaction methods for in situ molecular detection
WO2017186718A1 (fr) * 2016-04-25 2017-11-02 Universität Basel Édition d'allèles et ses applications
WO2017201476A1 (fr) 2016-05-20 2017-11-23 Regeneron Pharmaceuticals, Inc. Procédés pour briser la tolérance immunologique à l'aide de multiples arn de guidage
EP3604527A1 (fr) * 2016-06-02 2020-02-05 Sigma Aldrich Co. LLC Utilisation de protéines de liaison à l'adn programmables pour améliorer la modification ciblée du génome
EP3907286A1 (fr) * 2016-06-02 2021-11-10 Sigma-Aldrich Co., LLC Utilisation de protéines de liaison à l'adn programmables pour améliorer la modification ciblée du génome
US10266851B2 (en) 2016-06-02 2019-04-23 Sigma-Aldrich Co. Llc Using programmable DNA binding proteins to enhance targeted genome modification
EP3272867A1 (fr) * 2016-06-02 2018-01-24 Sigma-Aldrich Co. LLC Utilisation de protéines de liaison à l'adn programmables pour améliorer la modification ciblée du génome
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
EP4230648A2 (fr) 2016-07-28 2023-08-23 Regeneron Pharmaceuticals, Inc. Variants de gpr56 et leurs utilisations
WO2018022967A1 (fr) 2016-07-28 2018-02-01 Regeneron Pharmaceuticals, Inc. Variants de gpr156 et leurs utilisations
WO2018023014A1 (fr) 2016-07-29 2018-02-01 Regeneron Pharmaceuticals, Inc. Souris comprenant des mutations entraînant l'expression de la fibrilline 1 tronquée en c
US11566263B2 (en) 2016-08-02 2023-01-31 Editas Medicine, Inc. Compositions and methods for treating CEP290 associated disease
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11912987B2 (en) 2016-08-03 2024-02-27 KSQ Therapeutics, Inc. Methods for screening for cancer targets
US11078481B1 (en) 2016-08-03 2021-08-03 KSQ Therapeutics, Inc. Methods for screening for cancer targets
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11946163B2 (en) 2016-09-02 2024-04-02 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
US11078483B1 (en) 2016-09-02 2021-08-03 KSQ Therapeutics, Inc. Methods for measuring and improving CRISPR reagent function
WO2018054911A1 (fr) 2016-09-23 2018-03-29 Bayer Cropscience Nv Optimisation ciblée du génome dans des plantes
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
WO2018073237A1 (fr) 2016-10-17 2018-04-26 The University Court Of The University Of Edinburgh Porcs comprenant le gène cd163 modifié et procédés associés
US10912797B2 (en) 2016-10-18 2021-02-09 Intima Bioscience, Inc. Tumor infiltrating lymphocytes and methods of therapy
US11154574B2 (en) 2016-10-18 2021-10-26 Regents Of The University Of Minnesota Tumor infiltrating lymphocytes and methods of therapy
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11859219B1 (en) 2016-12-30 2024-01-02 Flagship Pioneering Innovations V, Inc. Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
WO2018136758A1 (fr) 2017-01-23 2018-07-26 Regeneron Pharmaceuticals, Inc. Variants de hsd17b13 et utilisations correspondantes
US11466271B2 (en) 2017-02-06 2022-10-11 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11851690B2 (en) 2017-03-14 2023-12-26 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11913015B2 (en) 2017-04-17 2024-02-27 University Of Maryland, College Park Embryonic cell cultures and methods of using the same
WO2018195545A2 (fr) 2017-04-21 2018-10-25 The General Hospital Corporation Variantes de cpf1 (cas12a) à spécificité pam modifiée
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
WO2018218166A1 (fr) 2017-05-25 2018-11-29 The General Hospital Corporation Utilisation de désaminases clivées pour limiter la désamination hors cible non désirée d'édition de bases
WO2018218206A1 (fr) 2017-05-25 2018-11-29 The General Hospital Corporation Architectures d'éditeur de base bipartite (bbe) et édition de doigt de zinc de type ii-c-cas9
WO2018226560A1 (fr) 2017-06-05 2018-12-13 Regeneron Pharmaceuticals, Inc. Variants de b4galt1 et utilisations associées
US10428319B2 (en) 2017-06-09 2019-10-01 Editas Medicine, Inc. Engineered Cas9 nucleases
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11814624B2 (en) 2017-06-15 2023-11-14 The Regents Of The University Of California Targeted non-viral DNA insertions
US11696572B2 (en) 2017-06-27 2023-07-11 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized ASGR1 locus
WO2019006034A1 (fr) 2017-06-27 2019-01-03 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus asgr1 humanisé
US11098325B2 (en) 2017-06-30 2021-08-24 Intima Bioscience, Inc. Adeno-associated viral vectors for gene therapy
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
WO2019028023A2 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour évaluer une disruption ou une excision médiée par crispr/cas et une recombinaison induite par crispr/cas à l'aide d'un acide nucléique donneur exogène in vivo
WO2019028029A1 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Évaluation de la recombinaison induite par crispr/cas avec un acide nucléique donneur exogène in vivo
WO2019028032A1 (fr) 2017-07-31 2019-02-07 Regeneron Pharmaceuticals, Inc. Cellules souches embryonnaires de souris transgéniques cas et souris et leurs utilisations
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
WO2019050948A1 (fr) * 2017-09-05 2019-03-14 Regeneron Pharmaceuticals, Inc. Administration d'un système d'édition génique comportant une seule particule rétrovirale et méthodes de génération et d'utilisation
WO2019050899A1 (fr) 2017-09-06 2019-03-14 Regeneron Pharmaceuticals, Inc. Variants apparentés au récepteur de l'interleukine 1 d'immunoglobuline unique (sigirr) et leurs utilisations
WO2019051033A1 (fr) 2017-09-07 2019-03-14 Regeneron Pharmaceuticals, Inc. Variants du membre 1 de la famille 14 des transporteurs de solutés (slc14a1) et leurs utilisations
WO2019067875A1 (fr) 2017-09-29 2019-04-04 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé et procédés d'utilisation
EP4276185A2 (fr) 2017-09-29 2023-11-15 Regeneron Pharmaceuticals, Inc. Rongeurs comprenant un locus ttr humanisé et procédés d'utilisation
WO2019079238A1 (fr) 2017-10-16 2019-04-25 Regeneron Pharmaceuticals, Inc. Variants de cornulin (crnn) et leurs utilisations
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11083753B1 (en) 2017-10-27 2021-08-10 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
US11033584B2 (en) 2017-10-27 2021-06-15 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
US11331346B2 (en) 2017-10-27 2022-05-17 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
US11590171B2 (en) 2017-10-27 2023-02-28 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
WO2019094735A1 (fr) 2017-11-10 2019-05-16 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant une mutation de slc30a8 et procédés d'utilisation
WO2019108983A1 (fr) 2017-11-30 2019-06-06 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus trkb humanisé
EP4299732A2 (fr) 2017-11-30 2024-01-03 Regeneron Pharmaceuticals, Inc. Rats comprenant un locus trkb humanisé
WO2019126578A1 (fr) * 2017-12-20 2019-06-27 Poseida Therapeutics, Inc. Compositions et procédés permettant de diriger des protéines vers des loci spécifiques dans le génome
WO2019123014A1 (fr) * 2017-12-22 2019-06-27 G+Flas Life Sciences Molécules d'ingénierie génomique chimère et procédés
US11293019B2 (en) 2017-12-22 2022-04-05 Gflas Life Sciences, Inc. Chimeric genome engineering molecules and methods
WO2019138083A1 (fr) 2018-01-12 2019-07-18 Basf Se Gène sous-jacent au nombre de qtl d'épillets par épi de blé sur le chromosome 7a
US10767193B2 (en) 2018-02-15 2020-09-08 Sigma-Aldrich Co. Llc Engineered CAS9 systems for eukaryotic genome modification
WO2019183123A1 (fr) 2018-03-19 2019-09-26 Regeneron Pharmaceuticals, Inc. Modulation de la transcription chez des animaux à l'aide de systèmes crispr/cas
US11434491B2 (en) 2018-04-19 2022-09-06 The Regents Of The University Of California Compositions and methods for gene editing
CN112105732A (zh) * 2018-05-10 2020-12-18 先正达参股股份有限公司 用于多核苷酸的靶向编辑的方法和组合物
EP3790976A4 (fr) * 2018-05-10 2022-08-10 Syngenta Participations Ag Procédés et compositions pour l'édition ciblée de polynucléotides
WO2019217803A1 (fr) 2018-05-10 2019-11-14 Auxolytic Ltd. Procédés et compositions de thérapie génique utilisant des cellules régulables auxotrophes
WO2019234754A1 (fr) 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Constructions d'acide nucléique et procédés d'utilisation de celles-ci
WO2019234750A1 (fr) 2018-06-07 2019-12-12 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Procédés de régénération et de transformation de cannabis
WO2020030783A2 (fr) 2018-08-10 2020-02-13 Vib Vzw Moyens et procédés de tolérance à la sécheresse dans des cultures
CN113166753A (zh) * 2018-08-21 2021-07-23 西格马-奥尔德里奇有限责任公司 胞质dna传感器途径的下调
WO2020041313A1 (fr) * 2018-08-21 2020-02-27 Sigma-Aldrich Co. Llc Régulation à la baisse de la voie de détection de l'adn cytosolique
WO2020056122A1 (fr) 2018-09-13 2020-03-19 Regeneron Pharmaceuticals, Inc. Rat à invalidation génique de facteur h de complément en tant que modèle de glomérulopathie c3
WO2020123377A1 (fr) 2018-12-10 2020-06-18 Neoimmunetech, Inc. Cellules défaillantes nrf-2 et utilisations de celles-ci
US11807878B2 (en) 2018-12-14 2023-11-07 Pioneer Hi-Bred International, Inc. CRISPR-Cas systems for genome editing
US10934536B2 (en) 2018-12-14 2021-03-02 Pioneer Hi-Bred International, Inc. CRISPR-CAS systems for genome editing
WO2020128478A1 (fr) 2018-12-19 2020-06-25 King's College London Procédés et compositions immunothérapeutiques
WO2020131632A1 (fr) 2018-12-20 2020-06-25 Regeneron Pharmaceuticals, Inc. Expansion de répétition à médiation par nucléase
WO2020163396A1 (fr) 2019-02-04 2020-08-13 The General Hospital Corporation Variants d'éditeur de base d'adn adénine avec édition d'arn hors cible réduite
US10947517B2 (en) 2019-02-15 2021-03-16 Sigma-Aldrich Co. Llc CRISPR/Cas fusion proteins and systems
WO2020169974A1 (fr) 2019-02-19 2020-08-27 King's College London Récepteurs antigéniques chimériques sensibles à l'hypoxie
EP4317950A2 (fr) 2019-03-18 2024-02-07 Regeneron Pharmaceuticals, Inc. Plate-forme de criblage crispr/cas pour identifier des modificateurs génétiques de l'ensemencement ou de l'agrégation de tau
WO2020190932A1 (fr) 2019-03-18 2020-09-24 Regeneron Pharmaceuticals, Inc. Plate-forme de criblage crispr/cas pour identifier des modificateurs génétiques de l'ensemencement ou de l'agrégation de la protéine tau
WO2020190927A1 (fr) 2019-03-18 2020-09-24 Regeneron Pharmaceuticals, Inc. Plate-forme de criblage crispr/cas pour révéler des vulnérabilités génétiques associées à une agrégation de tau
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
WO2020206162A1 (fr) 2019-04-03 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour l'insertion de séquences de codage d'anticorps dans un locus d'hébergement sûr
WO2020206134A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Procédés pour l'introduction sans cicatrice de modifications ciblées dans des vecteurs de ciblage
WO2020206139A1 (fr) 2019-04-04 2020-10-08 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus facteur 12 de coagulation humanisé
US11499164B2 (en) 2019-04-04 2022-11-15 Regeneran Pharmaceuticals, Inc. Methods for scarless introduction of targeted modifications into targeting vectors
US11111504B2 (en) 2019-04-04 2021-09-07 Regeneron Pharmaceuticals, Inc. Methods for scarless introduction of targeted modifications into targeting vectors
WO2020212541A1 (fr) 2019-04-16 2020-10-22 University Of Nottingham Souches fongiques, leur production et leurs utilisations
WO2020229241A1 (fr) 2019-05-10 2020-11-19 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
WO2020247452A1 (fr) 2019-06-04 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé ayant une mutation bêta-slip et procédés d'utilisation
WO2020247812A1 (fr) 2019-06-07 2020-12-10 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'albumine humanisé
WO2020252340A1 (fr) 2019-06-14 2020-12-17 Regeneron Pharmaceuticals, Inc. Modèles de tauopathie
WO2021050398A1 (fr) * 2019-09-10 2021-03-18 The Regents Of The University Of California Plate-forme de criblage de létalité synthétique pour cellules subissant un alt
WO2021048316A1 (fr) 2019-09-12 2021-03-18 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
WO2021050940A1 (fr) 2019-09-13 2021-03-18 Regeneron Pharmaceuticals, Inc. Modulation de la transcription chez des animaux à l'aide de systèmes crispr/cas administrés par des nanoparticules lipidiques
WO2021069387A1 (fr) 2019-10-07 2021-04-15 Basf Se Molécules d'acide nucléique régulatrices pour améliorer l'expression génique dans des plantes
WO2021092513A1 (fr) 2019-11-08 2021-05-14 Regeneron Pharmaceuticals, Inc. Stratégies crispr-vaa pour la thérapie du rétinoschisis juvénile lié à l'x
WO2021108363A1 (fr) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Régulation à la hausse médiée par crispr/cas d'un allèle ttr humanisé
WO2021110582A1 (fr) 2019-12-03 2021-06-10 Basf Se Molécules d'acides nucléiques régulatrices pour améliorer l'expression génique dans des plantes
WO2021154791A1 (fr) 2020-01-28 2021-08-05 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus pnpla3 humanisé et procédés d'utilisation
WO2021158883A1 (fr) 2020-02-07 2021-08-12 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus klkb1 humanisé et procédés d'utilisation
WO2021178556A1 (fr) 2020-03-04 2021-09-10 Regeneron Pharmaceuticals, Inc. Méthodes et compositions pour la sensibilisation de cellules tumorales à une thérapie immunitaire
WO2021195079A1 (fr) 2020-03-23 2021-09-30 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus ttr humanisé affichant une mutation v30m et méthodes d'utilisation
WO2021202938A1 (fr) 2020-04-03 2021-10-07 Creyon Bio, Inc. Apprentissage automatique fondé sur des oligonucléotides
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2021263146A2 (fr) 2020-06-26 2021-12-30 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus d'ace2 humanisé
WO2022120022A1 (fr) 2020-12-02 2022-06-09 Regeneron Pharmaceuticals, Inc. Lignées cellulaires de biocapteurs crispr sam et procédés d'utilisation associés
GB202103131D0 (en) 2021-03-05 2021-04-21 Biosystems Tech Limited Method for preparation of research organisms
WO2022240846A1 (fr) 2021-05-10 2022-11-17 Sqz Biotechnologies Company Méthodes pour administrer des molécules d'édition génomique au noyau ou au cytosol d'une cellule et leurs utilisations
WO2022251644A1 (fr) 2021-05-28 2022-12-01 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
WO2022256437A1 (fr) 2021-06-02 2022-12-08 Lyell Immunopharma, Inc. Cellules immunitaires déficientes en nr4a3 et leurs utilisations
US11884915B2 (en) 2021-09-10 2024-01-30 Agilent Technologies, Inc. Guide RNAs with chemical modification for prime editing
WO2023064924A1 (fr) 2021-10-14 2023-04-20 Codiak Biosciences, Inc. Cellules productrices modifiées pour la production de vésicules extracellulaires
WO2023077053A2 (fr) 2021-10-28 2023-05-04 Regeneron Pharmaceuticals, Inc. Procédés et compositions associés à crispr/cas pour la désactivation de c5
WO2023081847A1 (fr) 2021-11-04 2023-05-11 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus cacng1 modifié
WO2023108047A1 (fr) 2021-12-08 2023-06-15 Regeneron Pharmaceuticals, Inc. Modèle de maladie impliquant une myociline mutante et ses utilisations
GB202118058D0 (en) 2021-12-14 2022-01-26 Univ Warwick Methods to increase yields in crops
WO2023111541A1 (fr) 2021-12-14 2023-06-22 The University Of Warwick Procédés pour augmenter les rendements dans des cultures
EP4198124A1 (fr) 2021-12-15 2023-06-21 Versitech Limited Cas9-nucléases modifiées et leur procédé d'utilisation
WO2023122506A1 (fr) 2021-12-20 2023-06-29 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant des loci ace2 et tmprss humanisés
WO2023129974A1 (fr) 2021-12-29 2023-07-06 Bristol-Myers Squibb Company Génération de lignées de cellules avec site d'intégration
WO2023150181A1 (fr) 2022-02-01 2023-08-10 President And Fellows Of Harvard College Méthodes et compositions pour le traitement du cancer
WO2023150620A1 (fr) 2022-02-02 2023-08-10 Regeneron Pharmaceuticals, Inc. Insertion de transgène médiée par crispr dans des cellules néonatales
WO2023150798A1 (fr) 2022-02-07 2023-08-10 Regeneron Pharmaceuticals, Inc. Compositions et procédés permettant de définir des délais de traitement optimaux dans le cadre d'une maladie lysosomale
WO2023212677A2 (fr) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification de zones de sécurité extragéniques spécifiques de tissu pour des approches de thérapie génique
WO2023220603A1 (fr) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vecteurs et procédés de production d'anticorps in vivo
WO2023225665A1 (fr) 2022-05-19 2023-11-23 Lyell Immunopharma, Inc. Polynucléotides ciblant nr4a3 et leurs utilisations
WO2023235726A2 (fr) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Agents thérapeutiques d'interférence crispr pour une maladie d'expansion de répétition c9orf72
WO2023235725A2 (fr) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Agents thérapeutiques à base de crispr pour une maladie d'expansion de répétition c9orf72
WO2024003579A1 (fr) 2022-06-30 2024-01-04 University Of Newcastle Upon Tyne Prévention de la récurrence d'une maladie dans une thérapie de remplacement mitochondrial
WO2024026474A1 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions et méthodes d'administration médiée par le récepteur de la transferrine (tfr) au cerveau et au muscle
WO2024026488A2 (fr) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Animaux non humains comprenant un locus de récepteur de transférrine modifié
WO2024031053A1 (fr) 2022-08-05 2024-02-08 Regeneron Pharmaceuticals, Inc. Variants de tdp-43 résistants à l'agrégation
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024073606A1 (fr) 2022-09-28 2024-04-04 Regeneron Pharmaceuticals, Inc. Récepteurs modifiés résistants aux anticorps pour améliorer des thérapies à base de cellules

Also Published As

Publication number Publication date
IL238856B (en) 2018-03-29
CA2977152C (fr) 2019-04-09
CN105142669B (zh) 2018-07-03
AU2019201344A1 (en) 2019-03-21
EP2928496A4 (fr) 2017-03-01
LT3138911T (lt) 2019-02-25
DK3363902T3 (da) 2020-01-02
US20160298132A1 (en) 2016-10-13
KR20230070065A (ko) 2023-05-19
ES2714154T3 (es) 2019-05-27
US20170191082A1 (en) 2017-07-06
AU2020230246B2 (en) 2020-11-05
US20160298133A1 (en) 2016-10-13
PL3138912T3 (pl) 2019-04-30
IL300199A (en) 2023-03-01
DK3138912T3 (en) 2019-01-21
EP3617309A2 (fr) 2020-03-04
AU2017204031A1 (en) 2017-07-06
US20160298134A1 (en) 2016-10-13
AU2023216829A1 (en) 2023-09-14
US20210079427A1 (en) 2021-03-18
ES2653212T3 (es) 2018-02-06
AU2020230243A1 (en) 2020-10-01
EP3611263A1 (fr) 2020-02-19
PT3138911T (pt) 2018-12-28
EP3617309A3 (fr) 2020-05-06
AU2020273316A1 (en) 2020-12-17
AU2017204031B2 (en) 2018-06-14
DK3138911T3 (en) 2019-01-21
PT3138910T (pt) 2017-10-18
AU2013355214A1 (en) 2015-06-04
EP3360964B1 (fr) 2019-10-02
KR20210045515A (ko) 2021-04-26
KR20180011351A (ko) 2018-01-31
KR20150091052A (ko) 2015-08-07
PL3138911T3 (pl) 2019-04-30
ES2757808T3 (es) 2020-04-30
CN108913676A (zh) 2018-11-30
HK1218389A1 (zh) 2017-02-17
LT3138912T (lt) 2019-02-25
PT3360964T (pt) 2019-10-29
CA3034794A1 (fr) 2014-06-12
EP2928496A1 (fr) 2015-10-14
KR20190093680A (ko) 2019-08-09
SG11201503824SA (en) 2015-06-29
US20160298136A1 (en) 2016-10-13
IL257178A (en) 2018-03-29
KR102531576B1 (ko) 2023-05-11
US20160298138A1 (en) 2016-10-13
PL3138910T3 (pl) 2018-01-31
IL291129A (en) 2022-05-01
IL291129B2 (en) 2023-07-01
PL3360964T3 (pl) 2020-03-31
EP3360964A1 (fr) 2018-08-15
JP2021101706A (ja) 2021-07-15
AU2019201344C1 (en) 2020-12-24
SG10201910987SA (en) 2020-01-30
EP3138912B1 (fr) 2018-12-05
IL238856A0 (en) 2015-06-30
CN108913676B (zh) 2023-11-03
ES2713243T3 (es) 2019-05-20
EP3138910A1 (fr) 2017-03-08
BR112015012375A2 (pt) 2017-09-26
PT3138912T (pt) 2018-12-28
KR20200098727A (ko) 2020-08-20
EP2928496B1 (fr) 2019-10-09
SG10201800585VA (en) 2018-02-27
US20200140897A1 (en) 2020-05-07
AU2013355214B2 (en) 2017-06-15
US20210388396A1 (en) 2021-12-16
EP3138909A1 (fr) 2017-03-08
IL257178B (en) 2019-07-31
EP3363902B1 (fr) 2019-11-27
AU2020230243B2 (en) 2021-10-21
KR102145760B1 (ko) 2020-08-19
DK2928496T3 (da) 2019-11-11
SG10202107423UA (en) 2021-08-30
PT2928496T (pt) 2019-11-11
JP2016502840A (ja) 2016-02-01
PL3363902T3 (pl) 2020-05-18
JP2020120674A (ja) 2020-08-13
IL291129B1 (en) 2023-03-01
AU2018229489B2 (en) 2018-12-06
ES2769310T3 (es) 2020-06-25
ES2757325T3 (es) 2020-04-28
EP3135765A1 (fr) 2017-03-01
KR101844123B1 (ko) 2018-04-02
US10745716B2 (en) 2020-08-18
KR102243092B1 (ko) 2021-04-22
JP6620018B2 (ja) 2019-12-11
CN108715602A (zh) 2018-10-30
JP2019037231A (ja) 2019-03-14
US10731181B2 (en) 2020-08-04
IL267598A (en) 2019-08-29
KR20230003624A (ko) 2023-01-06
US20160298125A1 (en) 2016-10-13
KR102006880B1 (ko) 2019-08-02
AU2020230246A1 (en) 2020-10-01
DK3138910T3 (en) 2017-10-16
IL267598B (en) 2022-05-01
AU2022200330A1 (en) 2022-02-17
AU2022200330B2 (en) 2023-11-09
EP3363902A1 (fr) 2018-08-22
CA2891347C (fr) 2018-02-27
US20170073705A1 (en) 2017-03-16
LT3138910T (lt) 2017-11-10
EP3138910B1 (fr) 2017-09-20
PT3363902T (pt) 2019-12-19
US20160298137A1 (en) 2016-10-13
US20160298135A1 (en) 2016-10-13
JP2017192392A (ja) 2017-10-26
CN105142669A (zh) 2015-12-09
PL2928496T3 (pl) 2020-04-30
AU2018229489A1 (en) 2018-10-04
CA2891347A1 (fr) 2014-06-12
CA2977152A1 (fr) 2014-06-12
EP3141604A1 (fr) 2017-03-15
KR102479178B1 (ko) 2022-12-19
EP3138912A1 (fr) 2017-03-08
LT3363902T (lt) 2020-02-10
US20210207173A1 (en) 2021-07-08
DK3360964T3 (da) 2019-10-28
AU2020273316B2 (en) 2023-05-18
EP3138911A1 (fr) 2017-03-08
AU2019201344B2 (en) 2020-09-03
JP2022115994A (ja) 2022-08-09
EP3138911B1 (fr) 2018-12-05
US20160017366A1 (en) 2016-01-21

Similar Documents

Publication Publication Date Title
AU2019201344B2 (en) Crispr-based genome modification and regulation

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201380072477.4

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13859964

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2891347

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 238856

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 20157013843

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2013355214

Country of ref document: AU

Date of ref document: 20131205

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2015545838

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112015012375

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 2013859964

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 112015012375

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20150528